Low expression of miR7a exerts a biological function in testis.

MicroRNA-7: A miRNA with expanding roles in development and disease.

MicroRNAs (miRNAs) are a family of short, non-coding RNA molecules (∼22nt) involved in post-transcriptional control of gene expression. They act via base-pairing with mRNA transcripts that harbour target sequences, resulting in accelerated mRNA decay and/or translational attenuation. Given miRNAs mediate the expression of molecules involved in many aspects of normal cell development and functioning, it is not surprising that aberrant miRNA expression is closely associated with many human diseases. Their pivotal role in driving a range of normal cellular physiology as well as pathological processes has established miRNAs as potential therapeutics, as well as potential diagnostic and prognostic tools in human health. MicroRNA-7 (miR-7) is a highly conserved miRNA which displays restricted spatiotemporal expression during development and in maturity. In humans and mice, mature miR-7 is generated from three different genes, illustrating unexpected redundancy and also the importance of this miRNA in regulating key cellular processes. In this review we examine the expanding role of miR-7 in the context of health, with emphasis on organ differentiation and development, as well as in various mammalian diseases, particularly of the brain, heart, endocrine pancreas and skin, as well as in cancer. The more we learn about miR-7, the more we realise the complexity of its regulation and potential functional application both from a biomarker and therapeutic perspective.

Androgen regulation of micro-RNAs in prostate cancer.

BACKGROUND Androgens play a critical role in the growth of both androgen dependent and castration-resistant prostate cancer (CRPC). Only a few micro-RNAs (miRNAs) have been suggested to be androgen regulated. We aim to identify androgen regulated miRNAs. METHODS We utilized LNCaP derived model, we have established, and which overexpresses the androgen receptor (AR), the VCaP cell line, and 13 intact-castrated prostate cancer (PC) xenograft pairs, as well as clinical specimens of untreated (PC) and CRPC. The expression of miRNAs was analyzed by microarrays and quantitative RT-PCR (Q-RT-PCR). Transfection of pre-miR-141 and anti-miR-141 was also used. RESULTS Seventeen miRNAs were > 1.5-fold up- or downregulated upon dihydrotestosterone (DHT) treatment in the cell lines, and 42 after castration in the AR-positive xenografts. Only four miRNAs (miR-10a, miR-141, miR-150*, and miR-1225-5p) showed similar androgen regulation in both cell lines and xenografts. Of those, miR-141 was found to be expressed more in PC and CRPC compared to benign prostate hyperplasia. Additionally, the overexpression of miR-141 enhanced growth of parental LNCaP cells while inhibition of miR-141 by anti-miR-141 suppressed the growth of the LNCaP subline overexpressing AR. CONCLUSIONS Only a few miRNAs were found to be androgen-regulated in both cell lines and xenografts models. Of those, the expression of miR-141 was upregulated in cancer. The ectopic overexpression of miR-141 increased growth of LNCaP cell suggesting it may contribute to the progression of PC.

Degradation of Cellular miR-27 by a Novel, Highly Abundant Viral Transcript Is Important for Efficient Virus Replication In Vivo

Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3'-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3'-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3'-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.

Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a.

Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.

Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency.

Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPSC state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPSCs display similar characteristics to Oct4/Sox2/Klf4/Myc-iPSCs, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can cooperate in a powerful way to reprogram somatic cells to pluripotency.

Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA.

T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR 1 demonstrate that it directs degradation of mature miR-27 in a sequence-specific and binding-dependent manner. This viral strategy illustrates use of a ncRNA to manipulate host-cell gene expression via the miRNA pathway.

The epithelial inward rectifier channel Kir7.1 displays unusual K+ permeation properties.

Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+ channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis. In situ hybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed by in situ patch-clamp measurements). In cRNA-injected Xenopus oocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+ ([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of -100 mV, the inward current through Kir7.1 averaged -3.8 +/- 1.04 microA with 2 mM [K+]e and -4.82 +/- 1.87 microA with 96 mM [K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+ permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+ sensitivity was increased by a factor of approximately 25 (Ki = 27 microM). These findings support the important role of this site in the regulation of K+ permeability in Kir channels by extracellular cations.

Analysis of human cytomegalovirus-encoded microRNA activity during infection.

MicroRNAs (miRNAs) are expressed in a wide variety of organisms, ranging from plants to animals, and are key posttranscriptional regulators of gene expression. Virally encoded miRNAs are unique in that they could potentially target both viral and host genes. Indeed, we have previously demonstrated that a human cytomegalovirus (HCMV)-encoded miRNA, miR-UL112, downregulates the expression of a host immune gene, MICB. Remarkably, it was shown that the same miRNA also downregulates immediate-early viral genes and that its ectopic expression resulted in reduced viral replication and viral titers. The targets for most of the viral miRNAs, and hence their functions, are still unknown. Here we demonstrate that miR-UL112 also targets the UL114 gene, and we present evidence that the reduction of UL114 by miR-UL112 reduces its activity as uracil DNA glycosylase but only minimally affects virus growth. In addition, we show that two additional HCMV-encoded miRNAs, miR-US25-1 and miR-US25-2, reduce the viral replication and DNA synthesis not only of HCMV but also of other viruses, suggesting that these two miRNAs target cellular genes that are essential for virus growth. Thus, we suggest that in addition to miR-UL112, two additional HCMV miRNAs control the life cycle of the virus.

Expression of Versican 3′-Untranslated Region Modulates Endogenous MicroRNA Functions

BACKGROUND Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. METHODS AND FINDINGS Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. CONCLUSION Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

Pioneer factor Pax7 deploys a stable enhancer repertoire for specification of cell fate

Pioneer transcription factors establish new cell-fate competence by triggering chromatin remodeling. However, many features of pioneer action, such as their kinetics and stability, remain poorly defined. Here, we show that Pax7, by opening a unique repertoire of enhancers, is necessary and sufficient for specification of one pituitary lineage. Pax7 binds its targeted enhancers rapidly, but chromatin remodeling and gene activation are slower. Enhancers opened by Pax7 show a loss of DNA methylation and acquire stable epigenetic memory, as evidenced by binding of nonpioneer factors after Pax7 withdrawal. This work shows that transient Pax7 expression is sufficient for stable specification of cell identity. Analysis of Pax7 dynamics during pituitary lineage specification shows that Pax7 binds rapidly at uniquely marked heterochromatin pioneer sites and initiates chromatin opening that remains stable after Pax7 withdrawal, with loss of DNA hypermethylation at pioneered enhancers.

Epigenetic regulation of NANOG by miR-302 cluster-MBD2 completes induced pluripotent stem cell reprogramming.

While most somatic cells undergoing induced pluripotent stem (iPS) cell reprogramming with Yamanaka factors accumulate at stable partially reprogrammed stages, the molecular mechanisms required to achieve full reprogramming are unknown. MicroRNAs (miRNAs) fine-tune mRNA translation and are implicated in reprogramming, but miRNA functional targets critical for complete iPS cell reprogramming remain elusive. We identified methyl-DNA binding domain protein 2 (MBD2) as an epigenetic suppressor, blocking full reprogramming of somatic to iPS cells through direct binding to NANOG promoter elements preventing transcriptional activation. When we overexpressed miR-302 cluster we observed a significant increase in conversion of partial to fully reprogrammed iPS cells by suppressing MBD2 expression, thereby increasing NANOG expression. Thus, expression of exogenous miR-302 cluster (without miR-367) is efficient in attaining a fully reprogrammed iPS state in partially reprogrammed cells by relieving MBD2-mediated inhibition of NANOG expression. Our studies provide a direct molecular mechanism involved in generating complete human iPS cell reprogramming to study disease pathogenesis, drug screening, and for potential cell-based therapies.

Circular RNA and miR-7 in cancer.

MicroRNAs (miRNA) play important roles in fine-tuning gene expression and are often deregulated in cancer. The identification of competing endogenous RNA and circular RNA (circRNA) as important regulators of miRNA activity underscores the increasing complexity of ncRNA-mediated regulatory networks. Particularly, the recently identified circular RNA, ciRS-7, which acts as a designated miR-7 inhibitor/sponge, has conceptually changed the mechanistic understanding of miRNA networks. As miR-7 modulates the expression of several oncogenes, disclosing the regulation of miR-7 activity will likely advance the understanding of various cancer etiologies. Here, we review the current knowledge about the ciRS-7/miR-7 axis in cancer-related pathways and discuss possible models explaining the relevance of coexpressing miR-7 along with a circRNA inhibitor.

MicroRNAs can generate thresholds in target gene expression

MicroRNAs (miRNAs) are short, highly conserved noncoding RNA molecules that repress gene expression in a sequence-dependent manner. We performed single-cell measurements using quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We find that although the average level of repression is modest, in agreement with previous population-based measurements, the repression among individual cells varies dramatically. In particular, we show that regulation by miRNAs establishes a threshold level of target mRNA below which protein production is highly repressed. Near this threshold, protein expression responds sensitively to target mRNA input, consistent with a mathematical model of molecular titration. These results show that miRNAs can act both as a switch and as a fine-tuner of gene expression.

microRNA-29a suppresses cell proliferation by targeting SPARC in hepatocellular carcinoma.

In the present study, we constructed a lentivirus vector encoding the miR-29a             precursor and established two stably infected cell lines, PLC-29a and 97L-29a. The overexpression of miR-29a was confirmed by TaqMan RT-PCR and significantly             suppressed the growth of the hepatocellular carcinoma cell lines MHCC-97L and             PLC. Dual-luciferase reporter assays indicated that the SPARC mRNA 3'UTR was directly             targeted by miR-29a since the mutated 3'UTR was not affected. Silencing SPARC             expression by RNAi knockdown resulted in a similar effect as miR-29a overexpression             on hepatocellular carcinoma (HCC) cell growth regulation. Anti-miR-29a oligonucleotides             (AMOs) upregulated the levels of SPARC in the HCC cells. The phosphorylation of             AKT/mTOR downstream of SPARC was inhibited in miR-29a-overexpressing HCC cells. We further examined and compared the expression levels of miR-29a in HCC tissues             and the corresponding nearby non-cancerous liver tissues of 110 patients with             HCC by qRT-PCR, and significantly lower expression of miR-29a was observed in             the tissues affected by HCC. Our findings demonstrate that the expression of miR-29a             is important in the regulation of the SPARC-AKT pathway and HCC growth.

Long non-coding RNAs with low expression levels in cells are enriched in secreted exosomes.

Long non-coding RNAs (lncRNAs) are involved in regulating chromatin modifications, gene transcription, mRNA translation, and protein function. We recently reported a high variation in the basal expression levels of a panel of lncRNAs in HeLa and MCF-7 cells and their differential response to DNA damage induction. Here, we hypothesized that lncRNA molecules with different cellular expression may have a differential abundance in secreted exosomes, and their exosome levels would reflect cellular response to DNA damage. MALAT1, HOTAIR, lincRNA-p21, GAS5, TUG1, CCND1-ncRNA in exosomes secreted from cultured cells were characterized. A different expression pattern of lncRNAs in exosomes was seen compared to cells. RNA molecules with relative low expression levels (lincRNA-p21, HOTAIR, ncRNA-CCND1) were highly enriched in exosomes. TUG1 and GAS5 levels were moderately elevated in exosomes, whereas MALAT1--which was the most abundant molecule in cells--was present at levels comparable to its cellular levels. lincRNA-p21 and ncRNA-CCND1 were the main molecules; exosome levels of them best reflect the change of their cellular levels upon exposure of the cells to bleomycin-induced DNA damage. In conclusion, we provide evidence that lncRNAs have a differential abundance in exosomes, indicating a selective loading.

Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer

Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n=14), when compared with matched normal prostate (n=7). An expanded patient cohort (tumor n=149, matched normal n=30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio=2.005, 95% confidence interval (1.109, 3.625), P=0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.

Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells

BACKGROUND microRNAs (miRNAs) stably exist in circulating blood and are encapsulated in extracellular vesicles such as exosomes. The aims of this study were to identify which exosomal miRNAs are highly produced from epithelial ovarian cancer (EOC) cells, to analyze whether serum miRNA can be used to discriminate patients with EOC from healthy volunteers, and to investigate the functional role of exosomal miRNAs in ovarian cancer progression. METHODS Exosomes were collected from the culture media of serous ovarian cancer cell lines, namely TYK-nu and HeyA8 cells. An exosomal miRNA microarray revealed that several miRNAs including miR-99a-5p were specifically elevated in EOC-derived exosomes. Expression levels of serum miR-99a-5p in 62 patients with EOC, 26 patients with benign ovarian tumors, and 20 healthy volunteers were determined by miRNA quantitative reverse transcription-polymerase chain reaction. To investigate the role of exosomal miR-99a-5p in peritoneal dissemination, neighboring human peritoneal mesothelial cells (HPMCs) were treated with EOC-derived exosomes and then expression levels of miR-99a-5p were examined. Furthermore, mimics of miR-99a-5p were transfected into HPMCs and the effect of miR-99a-5p on cancer invasion was analyzed using a 3D culture model. Proteomic analysis with the tandem mass tag method was performed on HPMCs transfected with miR-99a-5p and then potential target genes of miR-99a-5p were examined. RESULTS The serum miR-99a-5p levels were significantly increased in patients with EOC, compared with those in benign tumor patients and healthy volunteers (1.7-fold and 2.8-fold, respectively). A receiver operating characteristic curve analysis showed with a cut-off of 1.41 showed sensitivity and specificity of 0.85 and 0.75, respectively, for detecting EOC (area under the curve = 0.88). Serum miR-99a-5p expression levels were significantly decreased after EOC surgeries (1.8 to 1.3, p = 0.002), indicating that miR-99a-5p reflects tumor burden. Treatment with EOC-derived exosomes significantly increased miR-99a-5p expression in HPMCs. HPMCs transfected with miR-99a-5p promoted ovarian cancer invasion and exhibited increased expression levels of fibronectin and vitronectin. CONCLUSIONS Serum miR-99a-5p is significantly elevated in ovarian cancer patients. Exosomal miR-99a-5p from EOC cells promotes cell invasion by affecting HPMCs through fibronectin and vitronectin upregulation and may serve as a target for inhibiting ovarian cancer progression.

Inhibition of SRC expression and activity inhibits tumor progression and metastasis of human pancreatic adenocarcinoma cells in an orthotopic nude mouse model.

The nonreceptor protein tyrosine kinase Src is overexpressed in 70% of pancreatic adenocarcinomas. Here, we describe the effect of molecular and pharmacological down-regulation of Src on incidence, growth, and metastasis of pancreatic tumor cells in an orthotopic model. Src expression in L3.6pl human pancreatic tumor cells was reduced by stable expression of a plasmid encoding small interfering RNA (siRNA) to c-src. In stable siRNA clones, Src expression was reduced >80%, with no change in expression of the related kinases c-Yes and c-Lyn, and proliferation rates were similar in all clones. Phosphorylation of Akt and p44/42 Erk mitogen-activated protein kinase and production of VEGF and IL-8 in culture supernatants were also reduced (P < 0.005). On orthotopic implantation of varying cell numbers into nude mice, tumor incidence was unchanged; however, in the siRNA clones, large tumors failed to develop, and incidence of metastasis was significantly reduced, suggesting that c-Src activity is critical to tumor progression. To examine this possibility further, animals bearing established wild-type tumors were treated with the Src/Abl-selective inhibitor BMS-354825 (dasatinib). Tumor size was decreased, and incidence of metastases was significantly reduced in treated mice compared with controls. These results demonstrate that Src activation contributes to pancreatic tumor progression in this model, offering Src as a candidate for targeted therapy.

Altered microRNA expression in bovine skeletal muscle with age.

Age-dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next-generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty-six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR-1, miR-206 and let-7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up-regulated and 22 miRNAs were down-regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta-mir-10020. A dual luciferase reporter assay was used to demonstrate that bta-mir-10020 directly targeted the 3'-UTR of the bovine ANGPT1 gene. The overexpression of bta-mir-10020 significantly decreased the DsRed fluorescence in the wild-type expression cassette compared to the mutant type. Using three computational approaches - miranda, pita and rnahybrid - these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type.

Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74.

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.

Functional screening identifies miRNAs inducing cardiac regeneration

In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers. Shortly after birth, however, cardiomyocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes. As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited. Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair. We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes. Two of these miRNAs (hsa-miR-590 and hsa-miR-199a) were further selected for testing and were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and to promote cardiomyocyte proliferation in both neonatal and adult animals. After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters. The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss.

MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Differentiation

The Polycomb Group (PcG) of chromatin modifiers regulates pluripotency and differentiation. Mammalian genomes encode multiple homologs of the Polycomb repressive complex 1 (PRC1) components, including five orthologs of the Drosophila Polycomb protein (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8). We have identified Cbx7 as the primary Polycomb ortholog of PRC1 complexes in embryonic stem cells (ESCs). The expression of Cbx7 is downregulated during ESC differentiation, preceding the upregulation of Cbx2, Cbx4, and Cbx8, which are directly repressed by Cbx7. Ectopic expression of Cbx7 inhibits differentiation and X chromosome inactivation and enhances ESC self-renewal. Conversely, Cbx7 knockdown induces differentiation and derepresses lineage-specific markers. In a functional screen, we identified the miR-125 and miR-181 families as regulators of Cbx7 that are induced during ESC differentiation. Ectopic expression of these miRNAs accelerates ESC differentiation via regulation of Cbx7. These observations establish a critical role for Cbx7 and its regulatory miRNAs in determining pluripotency.

Chromatin modifying enzymes as modulators of reprogramming

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

A Role for NuSAP in Linking Microtubules to Mitotic Chromosomes

The spindle apparatus is a microtubule (MT)-based machinery that attaches to and segregates the chromosomes during mitosis and meiosis. Self-organization of the spindle around chromatin involves the assembly of MTs, their attachment to the chromosomes, and their organization into a bipolar array. One regulator of spindle self-organization is RanGTP. RanGTP is generated at chromatin and activates a set of soluble, Ran-regulated spindle factors such as TPX2, NuMA, and NuSAP . How the spindle factors direct and attach MTs to the chromosomes are key open questions. Nucleolar and Spindle-Associated Protein (NuSAP) was recently identified as an essential MT-stabilizing and bundling protein that is enriched at the central part of the spindle . Here, we show by biochemical reconstitution that NuSAP efficiently adsorbs to isolated chromatin and DNA and that it can directly produce and retain high concentrations of MTs in the immediate vicinity of chromatin or DNA. Moreover, our data reveal that NuSAP-chromatin interaction is subject to Ran regulation and can be suppressed by Importin alpha (Impalpha) and Imp7. We propose that the presence of MT binding agents such as NuSAP, which can be directly immobilized on chromatin, are critical for targeting MT production to vertebrate chromosomes during spindle self-organization.

Synapsin I Cre transgene expression in male mice produces germline recombination in progeny.

The cre/LoxP system can produce conditional loss of gene function in specific cell types such as neurons. A transgenic mouse line, utilized by multiple studies, used the Synapsin I promoter to drive expression of cre (SynCre) to achieve neuronal-specific cre expression. Herein we describe that cre expression can also be observed in SynCre mice within the testes after being bred into a floxed transgenic mouse line. Cre transcript was expressed in testes resulting in recombination of the floxed substrate in testes. In the majority of cases, progeny of male SynCre mice inherited a germline recombined floxed allele, while this was never observed in progeny from female mice carrying the SynCre allele. This observation should alert investigators to a potential confound using these mice and enables male germ cell "deletor" strategies.

The developmental potential of iPSCs is greatly influenced by reprogramming factor selection.

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance.

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

miR-375 inhibits autophagy and reduces viability of hepatocellular carcinoma cells under hypoxic conditions.

BACKGROUND & AIMS Tumor cells survive hypoxic conditions by inducing autophagy. We investigated the roles of microRNAs (miRNAs) in regulating autophagy of hepatocellular carcinoma (HCC) cells under hypoxic conditions. METHODS We used gain- and loss-of-function methods to evaluate the effect of miRNAs on autophagy in human HCC cell lines (Huh7 and Hep3B) under hypoxic conditions. Autophagy was quantified by immunoblot, immunofluoresence, and transmission electron microscopy analyses, and after incubation of cells with bafilomycin A1. We used a luciferase reporter assay to confirm associations between miRNAs and their targets. We analyzed growth of HCC xenograft tumors in nude mice. RESULTS miR-375 was down-regulated in HCC cells and tissues; it inhibited autophagy under hypoxic conditions by suppressing the conversion of LC3I to LC3II and thereby autophagic flux. The ability of miR-375 to inhibit autophagy was independent of its ability to regulate 3'-phosphoinositide-dependent protein kinase-1-AKT-mammalian target of rapamycin signaling, but instead involved suppression of ATG7, an autophagy-associated gene. miR-375 bound directly to a predicted site in the 3' untranslated region of ATG7. Up-regulating miR-375 or down-regulating ATG7 inhibited mitochondrial autophagy of HCC cells, reduced the elimination of damaged mitochondria under hypoxia, increased release of mitochondrial apoptotic proteins, and reduced viability of HCC cells. In mice, xenograft tumors that expressed miR-375 had fewer autophagic cells, larger areas of necrosis, and grew more slowly than tumors from HCC cells that expressed lower levels of miR-375. CONCLUSIONS miR-375 inhibits autophagy by reducing expression of ATG7 and impairs viability of HCC cells under hypoxic conditions in culture and in mice. miRNAs that inhibit autophagy of cancer cells might be developed as therapeutics.

Requirement for the ERI/DICER complex in endogenous RNA interference and sperm development in Caenorhabditis elegans.

Small regulatory RNAs are key regulators of gene expression. One class of small regulatory RNAs, termed the endogenous small interfering RNAs (endo siRNAs), is thought to negatively regulate cellular transcripts via an RNA interference (RNAi)-like mechanism termed endogenous RNAi (endo RNAi). A complex of proteins composed of ERI-1/3/5, RRF-3, and DICER (the ERI/DICER complex) mediates endo RNAi processes in Caenorhabditis elegans. We conducted a genetic screen to identify additional components of the endo RNAi machinery. Our screen recovered alleles of eri-9, which encodes a novel DICER-interacting protein, and a missense mutation within the helicase domain of DICER [DCR-1(G492R)]. ERI-9(-) and DCR-1(G492) animals exhibit defects in endo siRNA expression and a concomitant failure to regulate mRNAs that exhibit sequence homology to these endo siRNAs, indicating that ERI-9 and the DCR-1 helicase domain function in the C. elegans endo RNAi pathway. We define a subset of Eri mutant animals (including eri-1, rrf-3, eri-3, and dcr-1, but not eri-9 or ergo-1) that exhibit temperature-sensitive, sperm-specific sterility and defects in X chromosome segregation. Among these mutants we find multiple aberrations in sperm development beginning with cytokinesis and extending through terminal differentiation. These results identify novel components of the endo RNAi machinery, demonstrate differential requirements for the Eri factors in the sperm-producing germline, and begin to delineate the functional requirement for the ERI/DICER complex in sperm development.

Functional proteomics identifies miRNAs to target a p27/Myc/phospho-Rb signature in breast and ovarian cancer

The myc oncogene is overexpressed in almost half of all breast and ovarian cancers, but attempts at therapeutic interventions against myc have proven to be challenging. Myc regulates multiple biological processes, including the cell cycle, and as such is associated with cell proliferation and tumor progression. We identified a protein signature of high myc, low p27 and high phospho-Rb significantly correlated with poor patient survival in breast and ovarian cancers. Screening of a miRNA library by functional proteomics in multiple cell lines and integration of data from patient tumors revealed a panel of five microRNAs (miRNAs) (miR-124, miR-365, miR-34b*, miR-18a and miR-506) as potential tumor suppressors capable of reversing the p27/myc/phospho-Rb protein signature. Mechanistic studies revealed an RNA-activation function of miR-124 resulting in direct induction of p27 protein levels by binding to and inducing transcription on the p27 promoter region leading to a subsequent G1 arrest. Additionally, in vivo studies utilizing a xenograft model demonstrated that nanoparticle-mediated delivery of miR-124 could reduce tumor growth and sensitize cells to etoposide, suggesting a clinical application of miRNAs as therapeutics to target the functional effect of myc on tumor growth.

Circulating plasma microRNAs as a screening method for detection of colorectal adenomas.

BACKGROUND MicroRNAs (miRNAs) are small non-coding RNA molecules. Reduced or increased levels of specific miRNAs are observed in colon and other cancers, supporting their role in carcinogenesis. Detection of colorectal polyps is the cornerstone of the Bowel Cancer Screening Programme in the UK. However, uptake of screening nationally remains under 60%. We aimed to see whether circulating plasma miRNAs can be used to screen for patients with colorectal polyps, adenomas, or both. METHODS Blood samples were taken from patients from the Bowel Cancer Screening Programme (asymptomatic but faecal occult blood testing [FOBt] positive). Plasma RNA was extracted, target miRNAs (19a, 98, 146b, 186, 191, 222*, 331-5p, 452, 625, 664, 1247) were identified on pooled case miRNA assay cards, and miRNA fraction was quantified by quantitative RT-PCR assay. Results were compared with endoscopy reports and with histology of any polyps identified and removed. Analysis was done with Excel (2011) and SPSS (version 20) software. FINDINGS 210 patients were included (117 with polyps, 12 with cancer, 81 healthy controls [FOBt positive]). The miRNA panel showed significant differences in expression (on t testing) for patients compared with controls for those with polyps, cancer, or both (miR-19a, p=0·0184; miR-98, p=0·0206; miR-146b, p=0·0029; miR-186, p=0·0006; miR-62,5 p=0·0008), polyps (miR-19a, p=0·0233; miR-98, p=0·0224; miR-146b, p=0·003; miR-186, p=0·0004; miR-625, p=0·001), adenomas (miR-19a, p=0·0339; miR-98, p=0·0266; miR-146b, p=0·0045; miR-186, p=0·0008; miR-625, p=0·0049), multiple adenomas (both sides of colon; miR-146b, p=0·0194; miR-186, p=0·0226; miR-625, p=0·0013), and right-sided adenomas (miR-98, p=0·031; miR-146b, p=0·0076; miR-186, p=0·0041; miR-331-5p, p=0·0142; miR-625, p=0·0049). Receiver operating characteristic analysis showed sensitivity of 60% or more, and specificity of 86% or more for men with polyps, men with adenomas, all patients with haemorrhoids or diverticulosis and polyps, and all patients with haemorrhoids or diverticulosis and adenomas. INTERPRETATION The target miRNAs that we identified showed significant differences in expression levels for patients with polyps and patients with adenomas from controls. Use of this panel has potential as a screening test. FUNDING Bowel Disease Research Foundation.

MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways

The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.

Dual Regulation of miRNA Biogenesis Generates Target Specificity in Neurotrophin-Induced Protein Synthesis

Control of translation is a fundamental source of regulation in gene expression. The induction of protein synthesis by brain-derived neurotrophic factor (BDNF) critically contributes to enduring modifications of synaptic function, but how BDNF selectively affects only a minority of expressed mRNAs is poorly understood. We report that BDNF rapidly elevates Dicer, increasing mature miRNA levels and inducing RNA processing bodies in neurons. BDNF also rapidly induces Lin28, causing selective loss of Lin28-regulated miRNAs and a corresponding upregulation in translation of their target mRNAs. Binding sites for Lin28-regulated miRNAs are necessary and sufficient to confer BDNF responsiveness to a transcript. Lin28 deficiency, or expression of a Lin28-resistant Let-7 precursor miRNA, inhibits BDNF translation specificity and BDNF-dependent dendrite arborization. Our data establish that specificity in BDNF-regulated translation depends upon a two-part posttranscriptional control of miRNA biogenesis that generally enhances mRNA repression in association with GW182 while selectively derepressing and increasing translation of specific mRNAs.

Polycomb Associates Genome-wide with a Specific RNA Polymerase II Variant, and Regulates Metabolic Genes in ESCs

Polycomb repressor complexes (PRCs) are important chromatin modifiers fundamentally implicated in pluripotency and cancer. Polycomb silencing in embryonic stem cells (ESCs) can be accompanied by active chromatin and primed RNA polymerase II (RNAPII), but the relationship between PRCs and RNAPII remains unclear genome-wide. We mapped PRC repression markers and four RNAPII states in ESCs using ChIP-seq, and found that PRC targets exhibit a range of RNAPII variants. First, developmental PRC targets are bound by unproductive RNAPII (S5p(+)S7p(-)S2p(-)) genome-wide. Sequential ChIP, Ring1B depletion, and genome-wide correlations show that PRCs and RNAPII-S5p physically bind to the same chromatin and functionally synergize. Second, we identify a cohort of genes marked by PRC and elongating RNAPII (S5p(+)S7p(+)S2p(+)); they produce mRNA and protein, and their expression increases upon PRC1 knockdown. We show that this group of PRC targets switches between active and PRC-repressed states within the ESC population, and that many have roles in metabolism.

Regulation of embryonic stem cell pluripotency by heat shock protein 90.

Deciphering the molecular basis of stem cell pluripotency is fundamental to the understanding of stem cell biology, early embryonic development, and to the clinical application of regenerative medicine. We report here that the molecular chaperone heat shock protein 90 (Hsp90) is essential for mouse embryonic stem cell (ESC) pluripotency through regulating multiple pluripotency factors, including Oct4, Nanog, and signal transducer and activator of transcription 3. Inhibition of Hsp90 by either 17-N-Allylamino-17-demethoxygeldanamycin or miRNA led to ESC differentiation. Overexpression of Hsp90β partially rescued the phenotype; in particular, the levels of Oct4 and Nanog were restored. Notably, Hsp90 associated with Oct4 and Nanog in the same cellular complex and protected them from degradation by the ubiquitin proteasome pathway, suggesting that Oct4 and Nanog are potential novel Hsp90 client proteins. In addition, Hsp90 inhibition reduced the mRNA level of Oct4, but not that of Nanog, indicating that Hsp90 participates in Oct4 mRNA processing or maturation. Hsp90 inhibition also increased expression of some protein markers for mesodermal lineages, implying that Hsp90 suppresses mesodermal differentiation from ESCs. These findings support a new role for Hsp90 in maintaining ESC pluripotency by sustaining the level of multiple pluripotency factors, particularly Oct4 and Nanog.

Interactions between SRY and SOX genes in mammalian sex determination.

The SRY gene on the mammalian Y chromosome undoubtedly acts to determine testis, but it is still quite unclear how. It was originally supposed that SRY acts directly to activate other genes in the testis-determining pathway. This paper presents an alternative hypothesis that SRY functions indirectly, by interacting with related genes SOX3 (from which SRY evolved) and SOX9 (which appears to be intimately involved in vertebrate gonad differentiation). Specifically, I propose that in females SOX3 inhibits SOX9 function, but in males, SRY inhibits SOX3 and permits SOX9 to enact its testis-determining role. This hypothesis makes testable predictions of the phenotypes of XX and XY individuals with deficiencies or overproduction of any of the three genes, and is able to account for the difficult cases of XX(SRY-) males and transdifferentiation in the absence of SRY. The hypothesis also suggests a way that the dominant SRY sex-determining system of present-day mammals may have evolved from an ancient system relying on SOX3 dosage.

Sperm chromatin proteomics identifies evolutionarily conserved fertility factors

Male infertility is a long-standing enigma of significant medical concern. The integrity of sperm chromatin is a clinical indicator of male fertility and in vitro fertilization potential: chromosome aneuploidy and DNA decondensation or damage are correlated with reproductive failure. Identifying conserved proteins important for sperm chromatin structure and packaging can reveal universal causes of infertility. Here we combine proteomics, cytology and functional analysis in Caenorhabditis elegans to identify spermatogenic chromatin-associated proteins that are important for fertility. Our strategy employed multiple steps: purification of chromatin from comparable meiotic cell types, namely those undergoing spermatogenesis or oogenesis; proteomic analysis by multidimensional protein identification technology (MudPIT) of factors that co-purify with chromatin; prioritization of sperm proteins based on abundance; and subtraction of common proteins to eliminate general chromatin and meiotic factors. Our approach reduced 1,099 proteins co-purified with spermatogenic chromatin, currently the most extensive catalogue, to 132 proteins for functional analysis. Reduction of gene function through RNA interference coupled with protein localization studies revealed conserved spermatogenesis-specific proteins vital for DNA compaction, chromosome segregation, and fertility. Unexpected roles in spermatogenesis were also detected for factors involved in other processes. Our strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7/19) cause male sterility. Our list therefore provides significant opportunity to identify causes of male infertility and targets for male contraceptives.

Endocrine control of spermatogenesis: Role of FSH and LH/ testosterone.

Evaluation of testicular functions (production of sperm and androgens) is an important aspect of preclinical safety assessment and testicular toxicity is comparatively far more common than ovarian toxicity. This chapter focuses (1) on the histological sequelae of disturbed reproductive endocrinology in rat, dog and nonhuman primates and (2) provides a review of our current understanding of the roles of gonadotropins and androgens. The response of the rodent testis to endocrine disturbances is clearly different from that of dog and primates with different germ cell types and spermatogenic stages being affected initially and also that the end-stage spermatogenic involution is more pronounced in dog and primates compared to rodents. Luteinizing hormone (LH)/testosterone and follicle-stimulating hormone (FSH) are the pivotal endocrine factors controlling testicular functions. The relative importance of either hormone is somewhat different between rodents and primates. Generally, however, both LH/testosterone and FSH are necessary for quantitatively normal spermatogenesis, at least in non-seasonal species.

A microRNA feedback loop regulates global microRNA abundance during aging.

Expression levels of many microRNAs (miRNAs) change during aging, notably declining globally in a number of organisms and tissues across taxa. However, little is known about the mechanisms or the biological relevance for this change. We investigated the network of genes that controls miRNA transcription and processing during C. elegans aging. We found that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. In particular, miR-71, known to influence life span and itself up-regulated during aging, represses alg-1/Argonaute expression post-transcriptionally during aging. Increased ALG-1 abundance in mir-71 loss-of-function mutants led to globally increased miRNA expression. Interestingly, these mutants demonstrated widespread mRNA expression dysregulation and diminished levels of variability both in gene expression and in overall life span. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organism's life may be partially explained by a miRNA-directed mechanism of age-associated decline.

miR-294/miR-302 promotes proliferation, suppresses G1-S restriction point, and inhibits ESC differentiation through separable mechanisms.

The miR-294 and miR-302 microRNAs promote the abbreviated G1 phase of the embryonic stem cell (ESC) cell cycle and suppress differentiation induced by let-7. Here, we evaluated the role of the retinoblastoma (Rb) family proteins in these settings. Under normal growth conditions, miR-294 promoted the rapid G1-S transition independent of the Rb family. In contrast, miR-294 suppressed the further accumulation of cells in G1 in response to nutrient deprivation and cell-cell contact in an Rb-dependent fashion. We uncovered five additional miRNAs (miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218) that silenced ESC self-renewal in the absence of other miRNAs, all of which were antagonized by miR-294 and miR-302. Four of the six differentiation-inducing miRNAs induced an Rb-dependent G1 accumulation. However, all six still silenced self-renewal in the absence of the Rb proteins. These results show that the miR-294/miR-302 family acts through Rb-dependent and -independent pathways to regulate the G1 restriction point and the silencing of self-renewal, respectively.

Methylation of histone H3 lysine-79 by Dot1p plays multiple roles in the response to UV damage in Saccharomyces cerevisiae.

Various proteins have been found to play roles in both the repair of UV damaged DNA and heterochromatin-mediated silencing in the yeast Saccharomyces cerevisiae. In particular, factors that are involved in the methylation of lysine-79 of histone H3 by Dot1p have been implicated in both processes, suggesting a bipartite function for this modification. We find that a dot1 null mutation and a histone H3 point mutation at lysine-79 cause increased sensitivity to UV radiation, suggesting that lysine-79 methylation is important for efficient repair of UV damage. Epistasis analysis between dot1 and various UV repair genes indicates that lysine-79 methylation plays overlapping roles within the nucleotide excision, post-replication and recombination repair pathways, as well as RAD9-mediated checkpoint function. In contrast, epistasis analysis with the H3 lysine-79 point mutation indicates that the lysine-to-glutamic acid substitution exerts specific effects within the nucleotide excision repair and post-replication repair pathways, suggesting that this allele only disrupts a subset of the functions of lysine-79 methylation. The overall results indicate the existence of distinct and separable roles of histone H3 lysine-79 methylation in the response to UV damage, potentially serving to coordinate the various repair processes.

MicroRNA Response of Primary Human Macrophages to Arcobacter Butzleri Infection

The role of microRNAs (miRNAs) in infectious diseases is becoming more and more apparent, and the use of miRNAs as a diagnostic tool and their therapeutic application has become the major focus of investigation. The aim of this study was to identify miRNAs involved in the immune signaling of macrophages in response to Arcobacter (A.) butzleri infection, an emerging foodborne pathogen causing gastroenteritis. Therefore, primary human macrophages were isolated and infected, and miRNA expression was studied by means of RNAseq. Analysis of the data revealed the expression of several miRNAs, which were previously associated with bacterial infections such as miR-155, miR-125, and miR-212. They were shown to play a key role in Toll-like receptor signaling where they act as fine-tuners to establish a balanced immune response. In addition, miRNAs which have yet not been identified during bacterial infections such as miR-3613, miR-2116, miR-671, miR-30d, and miR-629 were differentially regulated in A. butzleri-infected cells. Targets of these miRNAs accumulated in pathways such as apoptosis and endocytosis - processes that might be involved in A. butzleri pathogenesis. Our study contributes new findings about the interaction of A. butzleri with human innate immune cells helping to understand underlying regulatory mechanisms in macrophages during infection.

DNMT1 Inhibition Reprograms Pancreatic Cancer Stem Cells via Upregulation of the miR-17-92 Cluster.

Pancreatic ductal adenocarcinoma (PDAC) and other carcinomas are hierarchically organized, with cancer stem cells (CSC) residing at the top of the hierarchy, where they drive tumor progression, metastasis, and chemoresistance. As CSC and non-CSC share an identical genetic background, we hypothesize that differences in epigenetics account for the striking functional differences between these two cell populations. Epigenetic mechanisms, such as DNA methylation, play an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the role of DNA methylation in pancreatic CSC is obscure. In this study, we investigated the genome-wide DNA methylation profile of PDAC CSC, and we determined the importance of DNA methyltransferases for CSC maintenance and tumorigenicity. Using high-throughput methylation analysis, we discovered that sorted CSCs have a higher level of DNA methylation, regardless of the heterogeneity or polyclonality of the CSC populations present in the tumors analyzed. Mechanistically, CSC expressed higher DNMT1 levels than non-CSC. Pharmacologic or genetic targeting of DNMT1 in CSCs reduced their self-renewal and in vivo tumorigenic potential, defining DNMT1 as a candidate CSC therapeutic target. The inhibitory effect we observed was mediated in part through epigenetic reactivation of previously silenced miRNAs, in particular the miR-17-92 cluster. Together, our findings indicate that DNA methylation plays an important role in CSC biology and also provide a rationale to develop epigenetic modulators to target CSC plasticity and improve the poor outcome of PDAC patients. Cancer Res; 76(15); 4546-58. ©2016 AACR.

Cholesteryl Ester Transfer Protein (CETP) Polymorphisms Affect mRNA Splicing, HDL Levels, and Sex-Dependent Cardiovascular Risk

Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex-dependent CETP splicing effects on cardiovascular risk by a mechanism independent of circulating HDL-C levels.

Functional elements within the dynein microtubule-binding domain

Dynein interacts with microtubules through an ATP-sensitive linkage mapped to a structurally complex region of the heavy chain following the fourth P-loop motif. Virtually nothing is known regarding how binding affinity is achieved and modulated during ATP hydrolysis. We have performed a detailed dissection of the microtubule contact site, using fragment expression, alanine substitution, and peptide competition. Our work identifies three clusters of amino acids important for the physical contact with microtubules; two of these fall within a region sharing sequence homology with MAP1B, the third in a region just downstream. Amino acid substitutions within any one of these regions can eliminate or weaken microtubule binding (KK3379, 80, E3385, K3387, K3397, KK3410,11, W3414, RKK3418-20, F3426, R3464, S3466, and K3467), suggesting that their activities are highly coordinated. A peptide that actively displaces MAP1B from microtubules perturbs dynein binding, supporting previous evidence for similar sites of interaction. We have also identified four amino acids whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, and C3469). These suggest that nucleotide-sensitive affinity may be locally controlled at the site of contact. Our work is the first detailed description of dynein-tubulin interactions and provides a framework for understanding how affinity is achieved and modulated.

Direct Upregulation of STAT3 by MicroRNA-551b-3p Deregulates Growth and Metastasis of Ovarian Cancer.

3q26.2 amplification in high-grade serous ovarian cancer leads to increased expression of mature microRNA miR551b-3p, which is associated with poor clinical outcome. Importantly, miR551b-3p contributes to resistance to apoptosis and increased survival and proliferation of cancer cells in vitro and in vivo. miR551b-3p upregulates STAT3 protein levels, and STAT3 is required for the effects of miR551b-3p on cell proliferation. Rather than decreasing levels of target mRNA as expected, we demonstrate that miR551b-3p binds a complementary sequence on the STAT3 promoter, recruiting RNA polymerase II and the TWIST1 transcription factor to activate STAT3 transcription, and thus directly upregulates STAT3 expression. Furthermore, anti-miR551b reduced STAT3 expression in ovarian cancer cells in vitro and in vivo and reduced ovarian cancer growth in vivo. Together, our data demonstrate a role for miR551b-3p in transcriptional activation. Thus, miR551b-3p represents a promising candidate biomarker and therapeutic target in ovarian cancer.

LIN28B induces neuroblastoma and enhances MYCN levels via let-7 suppression

LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B–let-7–MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.

Uridylation of miRNAs by HEN1 SUPPRESSOR1 in Arabidopsis

HEN1-mediated 2'-O-methylation has been shown to be a key mechanism to protect plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) as well as animal piwi-interacting RNAs (piRNAs) from degradation and 3' terminal uridylation [1-8]. However, enzymes uridylating unmethylated miRNAs, siRNAs, or piRNAs in hen1 are unknown. In this study, a genetic screen identified a second-site mutation hen1 suppressor1-2 (heso1-2) that partially suppresses the morphological phenotypes of the hypomorphic hen1-2 allele and the null hen1-1 allele in Arabidopsis. HESO1 encodes a terminal nucleotidyl transferase that prefers to add untemplated uridine to the 3' end of RNA, which is completely abolished by 2'-O-methylation. heso1-2 affects the profile of u-tailed miRNAs and siRNAs and increases the abundance of truncated and/or normal sized ones in hen1, which often results in increased total amount of miRNAs and siRNAs in hen1. In contrast, overexpressing HESO1 in hen1-2 causes more severe morphological defects and less accumulation of miRNAs. These results demonstrate that HESO1 is an enzyme uridylating unmethylated miRNAs and siRNAs in hen1. These observations also suggest that uridylation may destabilize unmethylated miRNAs through an unknown mechanism and compete with 3'-to-5' exoribonuclease activities in hen1. This study shall have implications on piRNA uridylation in hen1 in animals.

Epigenetic regulation of miR-184 by MBD1 governs neural stem cell proliferation and differentiation.

Methyl-CpG binding protein 1 (MBD1) regulates gene expression via a DNA methylation-mediated epigenetic mechanism. We have previously demonstrated that MBD1 deficiency impairs adult neural stem/progenitor cell (aNSC) differentiation and neurogenesis, but the underlying mechanism was unclear. Here, we show that MBD1 regulates the expression of several microRNAs in aNSCs and, specifically, that miR-184 is directly repressed by MBD1. High levels of miR-184 promoted proliferation but inhibited differentiation of aNSCs, whereas inhibition of miR-184 rescued the phenotypes associated with MBD1 deficiency. We further found that miR-184 regulates the expression of Numblike (Numbl), a known regulator of brain development, by binding to the 3'-UTR of Numbl mRNA and affecting its translation. Expression of exogenous Numbl could rescue the aNSC defects that result from either miR-184 overexpression or MBD1 deficiency. Therefore, MBD1, miR-184, and Numbl form a regulatory network that helps control the balance between proliferation and differentiation of aNSCs.

Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase.

Ubiquitination controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by enzyme type 3 (E3) ubiquitin ligases. E3 enzymes are responsible for substrate specificity and catalyze the formation of an isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin. MIR1 and MIR2 are two E3 ubiquitin ligases encoded by Kaposi's sarcoma-associated herpesvirus that mediate the ubiquitination of major histocompatibility complex class I (MHC I) molecules and subsequent internalization. Here, we found that MIR1, but not MIR2, promoted down-regulation of MHC I molecules lacking lysine residues in their intracytoplasmic domain. In the presence of MIR1, these MHC I molecules were ubiquitinated, and their association with ubiquitin was sensitive to beta2-mercaptoethanol, unlike lysine-ubiquitin bonds. This form of ubiquitination required a cysteine residue in the intracytoplasmic tail of MHC I molecules. An MHC I molecule containing a single cysteine residue in an artificial glycine and alanine intracytoplasmic domain was endocytosed and degraded in the presence of MIR1. Thus, ubiquitination can occur on proteins lacking accessible lysines or an accessible N terminus.

Wnt7a/Fzd7 Signalling Directly Activates the Akt/mTOR Anabolic Growth Pathway in Skeletal Muscle

Wnt7a signals through its receptor Fzd7 to activate the planar-cell-polarity pathway and drive the symmetric expansion of satellite stem cells resulting in enhanced repair of skeletal muscle. In differentiated myofibres, we observed that Wnt7a binding to Fzd7 directly activates the Akt/mTOR growth pathway, thereby inducing myofibre hypertrophy. Notably, the Fzd7 receptor complex was associated with Gα(s) and PI(3)K and these components were required for Wnt7a to activate the Akt/mTOR growth pathway in myotubes. Wnt7a-Fzd7 activation of this pathway was completely independent of IGF-receptor activation. Together, these experiments demonstrate that Wnt7a-Fzd7 activates distinct pathways at different developmental stages during myogenic lineage progression, and identify a non-canonical anabolic signalling pathway for Wnt7a and its receptor Fzd7 in skeletal muscle.

Arginase I: a limiting factor for nitric oxide and polyamine synthesis by activated macrophages?

Because arginase hydrolyzes arginine to produce ornithine and urea, it has the potential to regulate nitric oxide (NO) and polyamine synthesis. We tested whether expression of the cytosolic isoform of arginase (arginase I) was limiting for NO or polyamine production by activated RAW 264.7 macrophage cells. RAW 264.7 cells, stably transfected to overexpress arginase I or beta-galactosidase, were treated with interferon-gamma to induce type 2 NO synthase or with lipopolysaccharide or 8-bromo-cAMP (8-BrcAMP) to induce ornithine decarboxylase. Overexpression of arginase I had no effect on NO synthesis. In contrast, cells overexpressing arginase I produced twice as much putrescine after activation than did cells expressing beta-galactosidase. Cells overexpressing arginase I also produced more spermidine after treatment with 8-BrcAMP than did cells expressing beta-galactosidase. Thus endogenous levels of arginase I are limiting for polyamine synthesis, but not for NO synthesis, by activated macrophage cells. This study also demonstrates that it is possible to alter arginase I levels sufficiently to affect polyamine synthesis without affecting induced NO synthesis.

N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex.

Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

Kinetic Analysis Reveals the Fate of a MicroRNA following Target Regulation in Mammalian Cells

Considerable details about microRNA (miRNA) biogenesis and regulation have been uncovered, but little is known about the fate of the miRNA subsequent to target regulation. To gain insight into this process, we carried out kinetic analysis of a miRNA's turnover following termination of its biogenesis and during regulation of a target that is not subject to Ago2-mediated catalytic cleavage. By quantitating the number of molecules of the miRNA and its target in steady state and in the course of its decay, we found that each miRNA molecule was able to regulate at least two target transcripts, providing in vivo evidence that the miRNA is not irreversibly sequestered with its target and that the nonslicing pathway of miRNA regulation is multiple-turnover. Using deep sequencing, we further show that miRNA recycling is limited by target regulation, which promotes posttranscriptional modifications to the 3' end of the miRNA and accelerates the miRNA's rate of decay. These studies provide new insight into the efficiency of miRNA regulation that help to explain how a miRNA can regulate a vast number of transcripts and that identify one of the mechanisms that impart specificity to miRNA decay in mammalian cells.

miR-302 Is Required for Timing of Neural Differentiation, Neural Tube Closure, and Embryonic Viability

The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15, in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development.

NDRG1 promotes growth of hepatocellular carcinoma cells by directly interacting with GSK-3β and Nur77 to prevent β-catenin degradation

The N-myc downstream regulated gene 1 (NDRG1) is significantly associated with advanced tumor stages and poor survival of hepatocellular carcinoma (HCC), thereby implicating it as a potential target for HCC treatment. We aim to further understand its biological roles in hepatocarcinogenesis, as a means to exploit it for therapeutic purposes. By screening using the ProtoArray® Human Protein Microarrays, we identified glycogen synthase kinase 3β (GSK-3β) and the orphan nuclear receptor (Nur77) as potential interaction partners of NDRG1. These interactions were confirmed in HCC cell lines in vitro by co-immunoprecipitation; and co-localizations of NDRG1 with GSK-3β and Nur77 were observed by immunofluorescence staining. Additionally, high levels of NDRG1 competitively bind to GSK-3β and Nur77 to allow β-catenin to escape degradation, with consequent elevated levels of downstream oncogenic genes. In vivo, we consistently observed that NDRG1 suppression in HCC xenografts decreased β-catenin levels and its downstream target Cyclin D1, with concomitant tumor growth inhibition. Clinically, the over-expression of NDRG1 in HCC patient samples is positively correlated with GSK-3β-9ser (| R | = 0.28, p = 0.01), Nur77 (| R | = 0.42, p < 0.001), and β-catenin (| R |= 0.32, p = 0.003) expressions. In conclusion, we identified GSK-3β and Nur77 as novel interaction partners of NDRG1. These protein-protein interactions regulate the turnover of β-catenin and subsequent downstream signaling mediated by β-catenin in HCC cells, and provides potential targets for future therapeutic interventions.

MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets.

Small non-coding microRNAs (miRNAs) contribute to cancer development and progression, and are differentially expressed in normal tissues and cancers. However, the specific role of miRNAs in the metastatic process is still unknown. To seek a specific miRNA expression signature characterizing the metastatic phenotype of solid tumours, we performed a miRNA microarray analysis on 43 paired primary tumours (ten colon, ten bladder, 13 breast, and ten lung cancers) and one of their related metastatic lymph nodes. We identified a metastatic cancer miRNA signature comprising 15 overexpressed and 17 underexpressed miRNAs. Our results were confirmed by qRT-PCR analysis. Among the miRNAs identified, some have a well-characterized association with cancer progression, eg miR-10b, miR-21, miR-30a, miR-30e, miR-125b, miR-141, miR-200b, miR-200c, and miR-205. To further support our data, we performed an immunohistochemical analysis for three well-defined miRNA gene targets (PDCD4, DHFR, and HOXD10 genes) on a small series of paired colon, breast, and bladder cancers, and one of their metastatic lymph nodes. We found that the immunohistochemical expression of these targets significantly follows the corresponding miRNA deregulation. Our results suggest that specific miRNAs may be directly involved in cancer metastasis and that they may represent a novel diagnostic tool in the characterization of metastatic cancer gene targets.

MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression

Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression.

C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins

An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question, we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats, but not stop codon–interrupted “RNA-only” repeats in Drosophila caused adult-onset neurodegeneration. Thus, expanded repeats promoted neurodegeneration through dipeptide repeat proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence revealed that both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.

NuSAP is essential for chromatin-induced spindle formation during early embryogenesis.

Mitotic spindle assembly is mediated by two processes: a centrosomal and a chromosomal pathway. RanGTP regulates the latter process by releasing microtubule-associated proteins from inhibitory complexes. NuSAP, a microtubule- and DNA-binding protein, is a target of RanGTP and promotes the formation of microtubules near chromosomes. However, the contribution of NuSAP to cell proliferation in vivo is unknown. Here, we demonstrate that the expression of NuSAP highly correlates with cell proliferation during embryogenesis and adult life, making it a reliable marker of proliferating cells. Additionally, we show that NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. As a result of sustained spindle checkpoint activity, the cells are unable to progress through mitosis, eventually leading to caspase activation and apoptotic cell death. Together, our findings demonstrate that NuSAP is essential for proliferation of embryonic cells and, simultaneously, they underscore the importance of chromatin-induced spindle assembly.

The potential role of miRNAs 21 and 199-a in early diagnosis of hepatocellular carcinoma.

BACKGROUND Hepatocellular carcinoma (HCC) is regarded as one of the most common malignancies and among the leading causes of cancer death among the whole world. The most urgent needs are to find sensitive markers for early diagnosis for HCC. MicroRNAs (miRNAs) are reported as a group of small non-coding RNAs that can function as endogenous RNA interference to regulate expression of the targeted genes. This study was conducted to detect the serum and tissue expression of miR 21 and miR 199-a to be applied as early detectors for HCC. METHODS A total of 40 serum and tissue samples (17 samples from chronic hepatitis and 23 samples from HCC patients) were collected. The levels of the two mature miRNAs (miR-21 and miR-199-a) were detected by real time quantitative reverse-transcriptase PCR (RT-qPCR) in sera and tissues of chronic hepatitis and HCC patients. Besides, miR-21 and miR-199-a levels in relation to clinical and pathological factors were explored. RESULTS We found that the expression of serum miR-21 was distinctly increased in HCC compared with chronic hepatitis (P<0.001). miR 199-a was distinctly decreased in HCC compared with chronic hepatitis (P<0.001). In addition, median of miR 21 was increased in malignant when compared to adjacent non-malignant tissues without significant differences (P=0.191) while miR 199-a was significantly decreased in malignant when compared to adjacent nonmalignant tissues (P<0.001). ROC analysis showed that miR-21 and miR-199-a might be potential biomarkers for HCC. CONCLUSIONS In conclusion, the expression of miR-21 was significantly up-regulated and miR-199-a was significantly down regulated in serum of patients with HCC. Due to their reasonable sensitivity and specificity for disease progression, miR-21 and miR-199-a could be used as potential circulating biomarkers for HCC.

Expression of a human cytomegalovirus latency-associated homolog of interleukin-10 during the productive phase of infection.

The human cytomegalovirus UL111A region is active during both productive and latent phases of infection. During productive infection, the virus expresses ORF79, a protein with oncogenic properties, and cmvIL-10, a functional homolog of human IL-10. During latent infection of myeloid progenitor cells, an alternately spliced variant of cmvIL-10, termed latency-associated (LA) cmvIL-10 has previously been identified. To determine whether LAcmvIL-10 transcription occurs during productive infection, we performed 5' and 3' RACE to map UL111A-region transcripts in productively infected human foreskin fibroblasts (HFFs). This analysis revealed the presence of a singly spliced UL111A-region transcript predicted to encode LAcmvIL-10. This transcript was expressed in HFFs with early (beta) kinetics, a temporal class that differs from that of ORF79 (alpha kinetics) and cmvIL-10 (gamma kinetics). These data identify and map a transcript encoding a latency-associated homolog of IL-10 which is expressed by the virus during the productive phase of infection.

A SUMO-Dependent Protein Network Regulates Chromosome Congression during Oocyte Meiosis

During Caenorhabditis elegans oocyte meiosis, a multi-protein ring complex (RC) localized between homologous chromosomes, promotes chromosome congression through the action of the chromokinesin KLP-19. While some RC components are known, the mechanism of RC assembly has remained obscure. We show that SUMO E3 ligase GEI-17/PIAS is required for KLP-19 recruitment to the RC, and proteomic analysis identified KLP-19 as a SUMO substrate in vivo. In vitro analysis revealed that KLP-19 is efficiently sumoylated in a GEI-17-dependent manner, while GEI-17 undergoes extensive auto-sumoylation. GEI-17 and another RC component, the kinase BUB-1, contain functional SUMO interaction motifs (SIMs), allowing them to recruit SUMO modified proteins, including KLP-19, into the RC. Thus, dynamic SUMO modification and the presence of SIMs in RC components generate a SUMO-SIM network that facilitates assembly of the RC. Our results highlight the importance of SUMO-SIM networks in regulating the assembly of dynamic protein complexes.

ROR-γ drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer

The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoic acid receptor-related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits nuclear receptor coactivator 1 and 3 (NCOA1 and NCOA3, also known as SRC-1 and SRC-3) to an AR-ROR response element (RORE) to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of both AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR target gene network. Finally, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing, but not AR-negative, xenograft PCa models, and they effectively sensitized CRPC tumors to enzalutamide, without overt toxicity, in mice. Taken together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and as a potential therapeutic target for advanced PCa.

LincRNA-p21 suppresses target mRNA translation.

Mammalian long intergenic noncoding RNAs (lincRNAs) are best known for modulating transcription. Here we report a posttranscriptional function for lincRNA-p21 as a modulator of translation. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNA-p21, leading to lower lincRNA-p21 stability. Under reduced HuR levels, lincRNA-p21 accumulated in human cervical carcinoma HeLa cells, increasing its association with JUNB and CTNNB1 mRNAs and selectively lowering their translation. With elevated HuR, lincRNA-p21 levels declined, which in turn derepressed JunB and β-catenin translation and increased the levels of these proteins. We propose that HuR controls translation of a subset of target mRNAs by influencing lincRNA-p21 levels. Our findings uncover a role for lincRNA as a posttranscriptional inhibitor of translation.

Relationship between Sloan-Kettering virus expression and mouse follicular development

Sloan-Kettering virus gene product (Ski) is an unique nuclear pro-oncoprotein and belongs to the ski/sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. Ski/SnoN are important transcription regulators of the transforming growth factor-β (TGF-β) superfamily and function mainly through heterodimers. Since TGF-β superfamily are key regulators of follicle development and it has been previously shown that SnoN is also vital to follicle development, this research was conducted to clarify the relationship between Ski expression and mouse follicular development, in ovaries of neonatal and gonadotropin-induced immature mice by immunohistochemical and real-time PCR techniques. In postnatal mice, positive staining for Ski was highly detected in oocyte nuclei at postnatal day 1. With follicular development, the localization moved gradually from oocyte nuclei to perinuclear space and the total levels decreased. During the estrous cycle, Ski expression was apparent at proestrus and estrus, faint at metestrus, highest at diestrus. After injection of gonadotropin, Ski was found in perinuclear space and weak in oocyte nuclei. Following the initiation of luteinization, the expression of Ski was found in corpus luteum. Real-time PCR results also showed that Ski mRNA expression was opposite to ovulation-related genes during the cumulus expansion, with the development of the follicles, its expression level decreased. Ski is expressed in a specific manner during follicle development, ovulation and luteinization. So Ski might play essential roles in these processes especially during early follicular development.

LncRNA-GAS5 induces PTEN expression through inhibiting miR-103 in endometrial cancer cells

BACKGROUND Growth arrest-specific 5 (GAS5) was reported to be implicated and aberrantly express in multiple cancers. However, the expression and mechanism of action of GAS5 were largely poor understood in endometrial carcinoma. RESULTS According to the result of real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and flow cytometry analysis, we identified that GAS5 was down-regulated in endometrial cancer cells and stimulated the apoptosis of endometrial cancer cells. To investigate the expression of GAS5, PTEN and miR-103, RT-PCR was performed. And we found that the expression of PTEN was up-regulated when endometrial cancer cells overexpressed GAS5. The prediction of bioinformatics online revealed that GAS5 could bind to miR-103, which was further found to be regulated by GAS5. Finally, we found that miR-103 mimic could decrease the mRNA and protein levels of PTEN through luciferase reporter assay and western blotting, and GAS5 plasmid may reverse this regulation effect in endometrial cancer cells. CONCLUSION In summary, we demonstrate that GAS5 acts as an tumor suppressor lncRNA in endometrial cancer. Through inhibiting the expression of miR-103, GAS5 significantly enhanced the expression of PTEN to promote cancer cell apoptosis, and, thus, could be an important mediator in the pathogenesis of endometrial cancer.

Nuclear RNAi maintains heritable gene silencing in Caenorhabditis elegans.

RNA interference (RNAi) is heritable in Caenorhabditis elegans; the progeny of C. elegans exposed to dsRNA inherit the ability to silence genes that were targeted by RNAi in the previous generation. Here we investigate the mechanism of RNAi inheritance in C. elegans. We show that exposure of animals to dsRNA results in the heritable expression of siRNAs and the heritable deposition of histone 3 lysine 9 methylation (H3K9me) marks in progeny. siRNAs are detectable before the appearance of H3K9me marks, suggesting that chromatin marks are not directly inherited but, rather, reestablished in inheriting progeny. Interestingly, H3K9me marks appear more prominently in inheriting progeny than in animals directly exposed to dsRNA, suggesting that germ-line transmission of silencing signals may enhance the efficiency of siRNA-directed H3K9me. Finally, we show that the nuclear RNAi (Nrde) pathway maintains heritable RNAi silencing in C. elegans. The Argonaute (Ago) NRDE-3 associates with heritable siRNAs and, acting in conjunction with the nuclear RNAi factors NRDE-1, NRDE-2, and NRDE-4, promotes siRNA expression in inheriting progeny. These results demonstrate that siRNA expression is heritable in C. elegans and define an RNAi pathway that promotes the maintenance of RNAi silencing and siRNA expression in the progeny of animals exposed to dsRNA.

MicroRNAs: small RNAs with a big role in gene regulation

Key PointsMicroRNAs (miRNAs) are a family of ∼21–25-nucleotide small RNAs that negatively regulate gene expression at the post-transcriptional level. The founding members of the miRNA family, lin-4 and let-7, were identified through genetic screens for defects in the temporal regulation of Caenorhabditis elegans larval development. Owing to genome-wide cloning efforts, hundreds of miRNAs have now been identified in almost all metazoans, including flies, plants and mammals. MiRNAs exhibit temporally and spatially regulated expression patterns during diverse developmental and physiological processes. Most of the miRNAs that have been characterized so far seem to regulate aspects of development, including larval developmental transitions and neuronal development in C. elegans, growth control and apoptosis in Drosophila melanogaster, haematopoietic differentiation in mammals, and leaf development, flower development and embryogenesis in Arabidopsis thaliana. The majority of the animal miRNAs that have been characterized so far affect protein synthesis from their target mRNAs. On the other hand, most of the plant miRNAs studied so far direct the cleavage of their targets. The degree of complementarity between a miRNA and its target, at least in part, determines the regulatory mechanism. In animals, primary transcripts of miRNAs are processed sequentially by two RNase-III enzymes, Drosha and Dicer, into a small, imperfect dsRNA duplex (miRNA:miRNA*) that contains both the mature miRNA strand and its complementary strand (miRNA*). Relative instability at the 5′ end of the mature miRNA leads to the asymmetric assembly of the mature miRNA into the effector complex, the RNA-induced silencing complex (RISC).Ago proteins are a key component of the RISC. Multiple Ago homologues in various metazoan genomes indicate the existence of multiple RISCs that carry out related but specific biological functions. Bioinformatic prediction of miRNA targets has provided an important tool to explore the functions of miRNAs. However, the overall success rate of such predictions remains to be determined by experimental validation. AbstractMicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. The two founding members of the microRNA family were originally identified in Caenorhabditis elegans as genes that were required for the timed regulation of developmental events. Since then, hundreds of microRNAs have been identified in almost all metazoan genomes, including worms, flies, plants and mammals. MicroRNAs have diverse expression patterns and might regulate various developmental and physiological processes. Their discovery adds a new dimension to our understanding of complex gene regulatory networks.

Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue.

Stem cells generate many differentiated, short-lived cell types, such as blood, skin, and sperm, throughout adult life. Stem cells maintain a long-term capacity to divide, producing daughter cells that either self-renew or initiate differentiation. Although the surrounding microenvironment or "niche" influences stem cell fate decisions, few signals that emanate from the niche to specify stem cell self-renewal have been identified. Here we demonstrate that the apical hub cells in the Drosophila testis act as a cellular niche that supports stem cell self-renewal. Hub cells express the ligand Unpaired (Upd), which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in adjacent germ cells to specify self-renewal and continual maintenance of the germ line stem cell population.

FBW7 regulates endothelial functions by targeting KLF2 for ubiquitination and degradation

F-box and WD repeat domain-containing 7 (FBW7), the substrate-binding subunit of E3 ubiquitin ligase SCFFBW7 (a complex of SKP1, cullin-1 and FBW7), plays important roles in various physiological and pathological processes. Although FBW7 is required for vascular development, its function in the endothelium remains to be investigated. In this study, we show that FBW7 is an important regulator of endothelial functions, including angiogenesis, leukocyte adhesion and the endothelial barrier integrity. Using RNA interference, we found that the depletion of FBW7 markedly impairs angiogenesis in vitro and in vivo. We identified the zinc finger transcription factor Krüppel-like factor 2 (KLF2) as a physiological target of FBW7 in endothelial cells. Knockdown of FBW7 expression resulted in the accumulation of endogenous KLF2 protein in endothelial cells. FBW7-mediated KLF2 destruction was shown to depend on the phosphorylation of KLF2 via glycogen synthase kinase-3 (GSK3) at two conserved phosphodegrons. Mutating these phosphodegron motifs abolished the FBW7-mediated degradation and ubiquitination of KLF2. The siRNA-mediated knockdown of FBW7 showed that KLF2 is an essential target of FBW7 in the regulation of endothelial functions. Moreover, FBW7-mediated KLF2 degradation was shown to be critical for angiogenesis in teratomas and in zebrafish development. Taken together, our study suggests a role for FBW7 in the processes of endothelial cell migration, angiogenesis, inflammation and barrier integrity, and provides novel insights into the regulation of KLF2 stability in vivo.

The calcium sensor STIM1 is regulated by androgens in prostate stromal cells.

BACKGROUND Prostate development and maintenance in the adult results from an interaction of stromal and glandular components. Androgens can drive this process by direct action on the stroma. We investigated whether there was a direct link between androgens and another key regulator of stromal cells, intracellular Ca2+ ([Ca2+ ]i ). METHODS Prostate stromal cells were freshly obtained and cultures derived from patients with benign prostatic hyperplasia. Gene expression in dihydrotestosterone treated and untreated cells was compared using Affymetrix gene expression arrays and Ca2+ regulated features were identified by Gene Ontology (GO). Changes in [Ca2+]i were determined in Fluo-4 loaded cells. Androgen regulation was confirmed by chromatin immunoprecipitaion. RESULTS Stromal cell cultures were sorted for expression of integrin α1 β1 , which enriched for cells expressing the androgen receptor (AR). We identified key functional categories, within the androgen-induced gene expression signature, focusing on genes involved in calcium signaling. From this analysis, stromal interaction molecule-1 (STIM1) was identified as a significantly differentially expressed gene with four relevant associated GO terms. DNA sequence analysis showed that the promoter region of STIM1 contained putative androgen response element sequences in which AR binding ability of STIM1 was confirmed. Androgens directly regulated STIM1 expression and STIM1 effects on store-operated calcium entry were inhibited by STIM1 knock-down. Reduced STIM1 expression in prostate stromal cells led to a reduction in basal Ca2+ levels, the amount of Ca2+ released by thapsigargin and a reduction in store filling following TG-induced store depletion. CONCLUSIONS These results indicate that androgens modulate [Ca2+]i through the direct regulation of the STIM1 gene by AR binding to the STIM1 promoter.

Cytomegalovirus microRNAs Facilitate Persistent Virus Infection in Salivary Glands

Micro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets' messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a number of viruses, particularly of the herpesvirus family, have been shown to express their own miRNAs to control both viral and cellular transcripts. Although some targets of viral miRNAs are known, their function in a physiologically relevant infection remains to be elucidated. As such, no in vivo phenotype of a viral miRNA knock-out mutant has been described so far. Here, we report on the first functional phenotype of a miRNA knock-out virus in vivo. During subacute infection of a mutant mouse cytomegalovirus lacking two viral miRNAs, virus production is selectively reduced in salivary glands, an organ essential for virus persistence and horizontal transmission. This phenotype depends on several parameters including viral load and mouse genetic background, and is abolished by combined but not single depletion of natural killer (NK) and CD4+ T cells. Together, our results point towards a miRNA-based immunoevasion mechanism important for long-term virus persistence.

Post-transcriptional Wnt Signaling Governs Epididymal Sperm Maturation

The canonical Wnt signaling pathway is of paramount importance in development and disease. An emergent question is whether the upstream cascade of the canonical Wnt pathway has physiologically relevant roles beyond β-catenin-mediated transcription, which is difficult to study due to the pervasive role of this protein. Here, we show that transcriptionally silent spermatozoa respond to Wnt signals released from the epididymis and that mice mutant for the Wnt regulator Cyclin Y-like 1 are male sterile due to immotile and malformed spermatozoa. Post-transcriptional Wnt signaling impacts spermatozoa through GSK3 by (1) reducing global protein poly-ubiquitination to maintain protein homeostasis; (2) inhibiting septin 4 phosphorylation to establish a membrane diffusion barrier in the sperm tail; and (3) inhibiting protein phosphatase 1 to initiate sperm motility. The results indicate that Wnt signaling orchestrates a rich post-transcriptional sperm maturation program and invite revisiting transcription-independent Wnt signaling in somatic cells as well.

Nuclear HuR accumulation through phosphorylation by Cdk1.

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Here, we present evidence that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR was specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was linked to its decreased association with 14-3-3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14-3-3 and hindering its post-transcriptional function and anti-apoptotic influence.

The Viral and Cellular MicroRNA Targetome in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.

Behavioral effects of estrogen receptor gene disruption in male mice.

Gonadal steroid hormones regulate sexually dimorphic development of brain functions and behaviors. Their nuclear receptors offer the opportunity to relate molecular events in neurons to simple instinctive mammalian behaviors. We have determined the role of estrogen receptor (ER) activation by endogenous estrogen in the development of male-typical behaviors by the use of transgenic estrogen-receptor-deficient (ERKO) mice. Surprisingly, in spite of the fact that they are infertile, ERKO mice showed normal motivation to mount females but they achieved less intromissions and virtually no ejaculations. Aggressive behaviors were dramatically reduced and male-typical offensive attacks were rarely displayed by ERKO males. Moreover, ER gene disruption demasculinized open-field behaviors. In the brain, despite the evident loss of functional ER protein, the androgen-dependent system appears to be normally present in ERKO mice. Together, these findings indicate that ER gene expression during development plays a major role in the organization of male-typical aggressive and emotional behaviors in addition to simple sexual behaviors.

Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1

Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer.

The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions.

Proteasomes are cylindrical structures that function in multiple cellular processes by degrading a wide variety of cytosolic and nuclear proteins. Substrate access and product release from the enclosed catalytic chamber occurs through axial pores that are opened by activator complexes. Here, we report high-resolution structures of wild-type and mutant archaeal proteasomes bound to the activator PA26. These structures support the proposal that an ordered open conformation is required for proteolysis and that its formation can be triggered by outward displacement of surrounding residues. The structures and associated biochemical assays reveal the mechanism of binding, which involves an interaction between the PA26 C terminus and a conserved lysine. Surprisingly, biochemical observations implicate an equivalent interaction for the unrelated ATP-dependent activators PAN and PA700.

Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells.

We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.

Identification of short-lived long non-coding RNAs as surrogate indicators for chemical stress response.

Abiotic and biotic stressors in human cells are often a result of sudden and/or frequent changes in environmental factors. The molecular response to stress involves elaborate modulation of gene expression and is of homeostatic, ecological, and evolutionary importance. Although attention has primarily focused on signaling pathways and protein networks, long non-coding RNAs (ncRNAs) are increasingly involved in the molecular mechanisms associated with responses to cellular stresses. We identified six novel short-lived long ncRNAs (MIR22HG, GABPB-AS1, LINC00152, IDI2-AS1, SNHG15, and FLJ33630) that responded to chemical stressors (cisplatin, cycloheximide, and mercury (II) oxide) in HeLa Tet-off cells. Our results indicate that short-lived long ncRNAs respond to general and specific chemical stressors. The expression levels of the short-lived long ncRNAs were elevated because of prolonged decay rates in response to chemical stressors and interruption of RNA degradation pathways. We propose that these long ncRNAs have the potential to be surrogate indicators of cellular stress responses.

Elevated expression of miR-24-3p is a potentially adverse prognostic factor in colorectal adenocarcinoma.

OBJECTIVES Several miRNAs are aberrantly expressed in cancer. miR-24-3p is involved in cancer-related cellular processes, including cell cycle control, cell growth, proliferation, and apoptosis. In this study, we examined the potential diagnostic and prognostic significance of miR-24-3p expression in colorectal adenocarcinoma. DESIGN AND METHODS Total RNA was isolated from 182 colorectal adenocarcinoma specimens and 86 paired non-cancerous colorectal mucosae. After polyadenylation of 2μg total RNA and reverse transcription into first-strand cDNA using an oligo-dT-adapter primer, miR-24-3p expression was quantified using an in-house-developed reverse-transcription real-time quantitative PCR method, based on the SYBR Green chemistry. SNORD43 (RNU43) was used as a reference gene. RESULTS miR-24-3p levels do not significantly differ between colorectal adenocarcinoma and non-cancerous colorectal mucosae. Thus, miR-24-3p expression cannot be used for diagnostic purposes. However, high miR-24-3p expression predicts poor disease-free survival (DFS) and overall survival (OS) of colorectal adenocarcinoma patients. Multivariate Cox regression analysis confirmed that miR-24-3p overexpression is a significant predictor of relapse in colorectal adenocarcinoma and that its prognostic significance is independent of other established prognostic factors and treatment of patients. Of note, miR-24-3p overexpression retains its rather unfavorable prognostic value in the subgroup of patients with advanced yet locally restricted colorectal adenocarcinoma (T3) and in those without distant metastasis (M0). Moreover, miR-24-3p overexpression is a potentially unfavorable prognosticator for patients who were not treated with radiotherapy. CONCLUSIONS Strong expression of miR-24-3p predicts poor DFS and OS of colorectal adenocarcinoma patients, independently of clinicopathological parameters that are currently used for prognosis in this human malignancy.

OncomiR-196 promotes an invasive phenotype in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathway

BACKGROUND MicroRNA-196 (miR-196), which is highly up-regulated in oral cancer cells, has been reported to be aberrantly expressed in several cancers; however, the significance of miR-196 in oral cancer has not yet been addressed. METHODS Cellular functions in response to miR-196 modulation were examined, including cell growth, migration, invasion and radio/chemosensitivity. Algorithm-based studies were used to identify the regulatory target of miR-196. The miR-196 target gene and downstream molecular mechanisms were confirmed by RT-qPCR, western blot, luciferase reporter and confocal microscopy analyses. miR-196 expression was determined in paired cancer and adjacent normal tissues from oral cancer patients. RESULTS Both miR-196a and miR-196b were highly over-expressed in the cancer tissue and correlated with lymph node metastasis (P = 0.001 and P = 0.006, respectively). Functionally, miR-196 actively promoted cell migration and invasion without affecting cell growth. Mechanistically, miR-196 performed it's their function by inhibiting NME4 expression and further activating p-JNK, suppressing TIMP1, and augmenting MMP1/9. CONCLUSION miR-196 contributes to oral cancer by promoting cell migration and invasion. Clinically, miR-196a/b was significantly over-expressed in the cancer tissues and correlated with lymph node metastasis. Thus, our findings provide new knowledge of the underlying mechanism of cancer metastasis. miR-196 may serve as a promising marker for better oral cancer management.

Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming.

Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.

Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.

Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.

A coding-independent function of gene and pseudogene mRNAs regulates tumour biology

The canonical role of messenger RNA (mRNA) is to deliver protein-coding information to sites of protein synthesis. However, given that microRNAs bind to RNAs, we hypothesized that RNAs could possess a regulatory role that relies on their ability to compete for microRNA binding, independently of their protein-coding function. As a model for the protein-coding-independent role of RNAs, we describe the functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene PTENP1 and the critical consequences of this interaction. We find that PTENP1 is biologically active as it can regulate cellular levels of PTEN and exert a growth-suppressive role. We also show that the PTENP1 locus is selectively lost in human cancer. We extended our analysis to other cancer-related genes that possess pseudogenes, such as oncogenic KRAS. We also demonstrate that the transcripts of protein-coding genes such as PTEN are biologically active. These findings attribute a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs.

Endogenous miRNA sponge lincRNA-RoR regulates Oct4, Nanog, and Sox2 in human embryonic stem cell self-renewal.

The embryonic stem cell (ESC) transcriptional and epigenetic networks are controlled by a multilayer regulatory circuitry, including core transcription factors (TFs), posttranscriptional modifier microRNAs (miRNAs), and some other regulators. However, the role of large intergenic noncoding RNAs (lincRNAs) in this regulatory circuitry and their underlying mechanism remains undefined. Here, we demonstrate that a lincRNA, linc-RoR, may function as a key competing endogenous RNA to link the network of miRNAs and core TFs, e.g., Oct4, Sox2, and Nanog. We show that linc-RoR shares miRNA-response elements with these core TFs and that linc-RoR prevents these core TFs from miRNA-mediated suppression in self-renewing human ESC. We suggest that linc-RoR forms a feedback loop with core TFs and miRNAs to regulate ESC maintenance and differentiation. These results may provide insights into the functional interactions of the components of genetic networks during development and may lead to new therapies for many diseases.

CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control.

MicroRNAs (miRNAs) are a recently identified class of epigenetic elements consisting of small noncoding RNAs that bind to the 3' untranslated region of mRNAs and down-regulate their translation to protein. miRNAs play critical roles in many different cellular processes including metabolism, apoptosis, differentiation, and development. We found 33 miRNAs expressed in CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human bone marrow and mobilized human peripheral blood stem cell harvests. We then combined these data with human HSPC mRNA expression data and with miRNA-mRNA target predictions, into a previously undescribed miRNA:mRNA interaction database called the Transcriptome Interaction Database. The in silico predictions from the Transcriptome Interaction Database pointed to miRNA control of hematopoietic differentiation through translational control of mRNAs critical to hematopoiesis. From these predictions, we formulated a model for miRNA control of stages of hematopoiesis in which many of the genes specifying hematopoietic differentiation are expressed by HSPCs, but are held in check by miRNAs until differentiation occurs. We validated miRNA control of several of these target mRNAs by demonstrating that their translation in fact is decreased by miRNAs. Finally, we chose miRNA-155 for functional characterization in hematopoiesis, because we predicted that it would control both myelopoiesis and erythropoiesis. As predicted, miRNA-155 transduction greatly reduced both myeloid and erythroid colony formation of normal human HSPCs.

MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors.

Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-alpha 1 and -2 (Col1a1 and -2). Transforming growth factor beta1 (TGF-beta), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-beta increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, deltaEF1. TGF-beta treatment or short hairpin RNAs targeting deltaEF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-beta also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to deltaEF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-beta in MMC. TGF-beta treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3' UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with deltaEF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-beta and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-beta-induced Col1a2 expression by down-regulating E-box repressors.

Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5′ cap structures of higher eukaryote mRNAs have ribose 2′-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2′-O-methylation of mRNA remains elusive. Here we show that 2′-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2′-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2′-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2′-O-methylation of mRNA suggests that RNA modifications such as 2′-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

Structure of the Guanine Nucleotide Exchange Factor Sec7 Domain of Human Arno and Analysis of the Interaction with ARF GTPase

Sec7-related guanine nucleotide exchange factors (GEFs) initiate vesicle budding from the Golgi membrane surface by converting the GTPase ARF to a GTP-bound, membrane-associated form. Here we report the crystal structure of the catalytic Sec7 homology domain of Arno, a human GEF for ARF1, determined at 2.2 angstroms resolution. The Sec7 domain is an elongated, all-helical protein with a distinctive hydrophobic groove that is phylogenetically conserved. Structure-based mutagenesis identifies the groove and an adjacent conserved loop as the ARF-interacting surface. The sites of Sec7 domain interaction on ARF1 have subsequently been mapped, by protein footprinting experiments, to the switch 1 and switch 2 GTPase regions, leading to a model for the interaction between ARF GTPases and Sec7 domain exchange factors.

Urine microRNAs as biomarkers for bladder cancer: a diagnostic meta-analysis

BACKGROUND The diagnostic value of microRNA (miRNA) detection in patients with bladder cancer (BCa) is controversial. We performed a diagnostic meta-analysis to evaluate current evidence on the use of miRNA assays to diagnose BCa. METHODS We systematically searched PubMed, Embase, and Web of Science for studies published before March 31, 2015. The pooled sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and area under the curve (AUC) were calculated to evaluate the overall test performance. Subgroup analyses were used to explore the between-study heterogeneity. Deeks' funnel plot asymmetry test was used to test publication bias. We applied the software of RevMan 5.2 and Stata 11.0 to the meta-analysis. RESULTS A total of 23 studies from nine articles were included in the meta-analysis, with a total of 719 patients and 494 controls. The pooled sensitivity and specificity were 0.75 (95% confidence interval [CI], 0.68-0.80) and 0.75 (95% CI, 0.70-0.80), respectively. The pooled positive likelihood ratio was 3.03 (95% CI, 2.50-3.67); negative likelihood ratio was 0.33 (95% CI, 0.27-0.42); and diagnostic odds ratio was 9.07 (95% CI, 6.35-12.95). The pooled AUC was 0.81 (95% CI, 0.78-0.85). Subgroup analyses indicated that the multiple miRNAs assays and urine supernatant assays showed high accuracies in diagnosing BCa. CONCLUSION The miRNA assays may serve as potential noninvasive diagnostic tool for the detection of BCa. However, the clinical application of miRNA assays for BCa diagnosis still needs further validation by large prospective studies.

Microtubule sliding activity of a kinesin-8 promotes spindle assembly and spindle length control

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide-disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.

Notch plays a critical role in the transition from proliferation to differentiation in the epidermis and corneal epithelium. Furthermore, aberrant Notch signaling is a feature of diseases like psoriasis, eczema, nonmelanoma skin cancer, and melanoma where differentiation and proliferation are impaired. Whereas much is known about the downstream events following Notch signaling, factors responsible for negatively regulating Notch receptor signaling after ligand activation are incompletely understood. Notch can undergo hydroxylation by factor-inhibiting hypoxia-inducible factor 1 (FIH-1); however, the biological significance of this phenomenon is unclear. Here we show that FIH-1 expression is up-regulated in diseased epidermis and corneal epithelium. Elevating FIH-1 levels in primary human epidermal keratinocytes (HEKs) and human corneal epithelial keratinocytes (HCEKs) impairs differentiation in submerged cultures and in a "three-dimensional" organotypic raft model of human epidermis, in part, via a coordinate decrease in Notch signaling. Knockdown of FIH-1 enhances keratinocyte differentiation. Loss of FIH-1 in vivo increased Notch activity in the limbal epithelium, resulting in a more differentiated phenotype. microRNA-31 (miR-31) is an endogenous negative regulator of FIH-1 expression that results in keratinocyte differentiation, mediated by Notch activation. Ectopically expressing miR-31 in an undifferentiated corneal epithelial cell line promotes differentiation and recapitulates a corneal epithelium in a three-dimensional raft culture model. Our results define a previously unknown mechanism for keratinocyte fate decisions where Notch signaling potential is, in part, controlled through a miR-31/FIH-1 nexus.

Sperm function tests and fertility.

Traditionally, the diagnosis of male infertility has depended upon a descriptive evaluation of human semen with emphasis on the number of spermatozoa that are present in the ejaculate, their motility and their morphology. The fundamental tenet underlying this approach is that male fertility can be defined by reference to a threshold concentration of motile, morphologically normal spermatozoa that must be exceeded in order to achieve conception. Many independent studies have demonstrated that this fundamental concept is flawed and, in reality, it is not so much the absolute number of spermatozoa that determines fertility, but their functional competence. In the light of this conclusion, a range of in vitro tests have been developed to monitor various aspects of sperm function including their potential for movement, cervical mucus penetration, capacitation, zona recognition, the acrosome reaction and sperm-oocyte fusion. Such functional assays have been found to predict the fertilizing capacity of human spermatozoa in vitro and in vivo with some accuracy. Recent developments in this field include the introduction of tests to assess the degree to which human spermatozoa have suffered oxidative stress as well as the integrity of their nuclear and mitochondrial DNA. Such assessments not only yield information on the fertilizing capacity of human spermatozoa but also their ability to support normal embryonic development.

Summary

Steroid receptor RNA activator (SRA), the only known RNA coactivator, augments transactivation by nuclear receptors (NRs). We identified SLIRP (SRA stem-loop interacting RNA binding protein) binding to a functional substructure of SRA, STR7. SLIRP is expressed in normal and tumor tissues, contains an RNA recognition motif (RRM), represses NR transactivation in a SRA- and RRM-dependent manner, augments the effect of Tamoxifen, and modulates association of SRC-1 with SRA. SHARP, a RRM-containing corepressor, also binds STR7, augmenting repression with SLIRP. SLIRP colocalizes with SKIP (Chr14q24.3), another NR coregulator, and reduces SKIP-potentiated NR signaling. SLIRP is recruited to endogenous promoters (pS2 and metallothionein), the latter in a SRA-dependent manner, while NCoR promoter recruitment is dependent on SLIRP. The majority of the endogenous SLIRP resides in the mitochondria. Our data demonstrate that SLIRP modulates NR transactivation, suggest it may regulate mitochondrial function, and provide mechanistic insight into interactions between SRA, SLIRP, SRC-1, and NCoR.

KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer

BACKGROUND Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas. METHODS We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally. RESULTS Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity. CONCLUSION These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.

Conserved Histone Variant H2A.Z Protects Euchromatin from the Ectopic Spread of Silent Heterochromatin

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.

DNA Methylation mediated down-regulating of MicroRNA-33b and its role in gastric cancer

The discovery of microRNAs (miRNAs) provides a new and powerful tool for studying the mechanism, diagnosis and treatment of human cancers. Currently, down-regulation of tumor suppressive miRNAs by CpG island hypermethylation is emerging as a common hallmark of cancer. Here, we reported that the down-regulation of miR-33b was associated with pM stage of gastric cancer (GC) patients. Ectopic expression of miR-33b in HGC-27 and MGC-803 cells inhibited cell proliferation, migration and invasion, which might be due to miR-33b targeting oncogene c-Myc. Moreover, enhanced methylation level of the CpG island upstream of miR-33b in GC patients with down-regulated miR-33b was confirmed by methylation-specific PCR (MSP) amplification. Furthermore, re-introduction of miR-33b significantly suppressed tumorigenesis of GC cells in the nude mice. In conclusion, miR-33b acts as a tumor suppressor and hypermethylation of the CpG island upstream of miR-33b is responsible for its down-regulation in gastric cancer.

Functional coupling of RNAP II transcription to spliceosome assembly.

The pathway of gene expression in higher eukaryotes involves a highly complex network of physical and functional interactions among the different machines involved in each step of the pathway. Here we established an efficient in vitro system to determine how RNA polymerase II (RNAP II) transcription is functionally coupled to pre-mRNA splicing. Strikingly, our data show that nascent pre-messenger RNA (pre-mRNA) synthesized by RNAP II is immediately and quantitatively directed into the spliceosome assembly pathway. In contrast, nascent pre-mRNA synthesized by T7 RNA polymerase is quantitatively assembled into the nonspecific H complex, which consists of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and is inhibitory for spliceosome assembly. Consequently, RNAP II transcription results in a dramatic increase in both the kinetics of splicing and overall yield of spliced mRNA relative to that observed for T7 transcription. We conclude that RNAP II mediates the functional coupling of transcription to splicing by directing the nascent pre-mRNA into spliceosome assembly, thereby bypassing interaction of the pre-mRNA with the inhibitory hnRNP proteins.