Cardio-miRNAs and onco-miRNAs: circulating miRNA-based diagnostics for non-cancerous and cancerous diseases

Cardiovascular diseases and cancers are the leading causes of morbidity and mortality in the world. MicroRNAs (miRNAs) are short non-coding RNAs that primarily repress target mRNAs. Here, miR-24, miR-125b, miR-195, and miR-214 were selected as representative cardio-miRs that are upregulated in human heart failure. To bridge the gap between miRNA studies in cardiology and oncology, the targets and functions of these miRNAs in cardiovascular diseases and cancers will be reviewed. ACVR1B, BCL2, BIM, eNOS, FGFR3, JPH2, MEN1, MYC, p16, and ST7L are miR-24 targets that have been experimentally validated in human cells. ARID3B, BAK1, BCL2, BMPR1B, ERBB2, FGFR2, IL6R, MUC1, SITR7, Smoothened, STAT3, TET2, and TP53 are representative miR-125b targets. ACVR2A, BCL2, CCND1, E2F3, GLUT3, MYB, RAF1, VEGF, WEE1, and WNT7A are representative miR-195 targets. BCL2L2, ß-catenin, BIM, CADM1, EZH2, FGFR1, NRAS, PTEN, TP53, and TWIST1 are representative miR-214 targets. miR-125b is a good cardio-miR that protects cardiomyocytes; miR-195 is a bad cardio-miR that elicits cardiomyopathy and heart failure; miR-24 and miR-214 are bi-functional cardio-miRs. By contrast, miR-24, miR-125b, miR-195, and miR-214 function as oncogenic or tumor suppressor miRNAs in a cancer (sub)type-dependent manner. Circulating miR-24 is elevated in diabetes, breast cancer and lung cancer. Circulating miR-195 is elevated in acute myocardial infarction, breast cancer, prostate cancer and colorectal adenoma. Circulating miR-125b and miR-214 are elevated in some cancers. Cardio-miRs and onco-miRs bear some similarities in functions and circulation profiles. miRNAs regulate WNT, FGF, Hedgehog and other signaling cascades that are involved in orchestration of embryogenesis and homeostasis as well as pathogenesis of human diseases. Because circulating miRNA profiles are modulated by genetic and environmental factors and are dysregulated by genetic and epigenetic alterations in somatic cells, circulating miRNA association studies (CMASs) within several thousands of cases each for common non-cancerous diseases and major cancers are necessary for miRNA-based diagnostics.


INTRODUCTION
MicroRNAs (miRNAs) are short non-coding RNAs that primarily repress protein expression from target mRNAs with imperfect or perfect complementarity through mRNA degradation and translational inhibition or mRNA cleavage, respectively (Kasinski and Slack, 2011;van Rooij and Olson, 2012). For example, are anti-angiogenic miRNAs that repress VEGFA (VEGF) (Wang and Olson, 2009;Katoh, 2013b). miR-200 family members inhibit epithelial-tomesenchymal transition (EMT) and self-renewal of stem cells through repression of ZEB1/2 and BMI1, respectively (Katoh and Katoh, 2008;Oishi et al., 2012;Feng et al., 2014). miRNAs regulate a variety of cellular processes, such as stemness, proliferation, senescence, apoptosis, inflammatory cytokine production, EMT, metastasis and drug resistance. Cardiovascular diseases and cancers are the leading causes of morbidity and mortality in the world (Lozano et al., 2012). miR-NAs involved in heart diseases (Divakaran and Mann, 2008), vascular diseases (Wang and Olson, 2009) and cancers (Croce, 2009) are designated cardio-miRs, angio-miRs, and onco-miRs, respectively, and the same miRNA can function as a cardio-miR, angio-miR or onco-miR in a context-dependent manner. Among 32,233 miRNA manuscripts in the PubMed database, 3334 and 15,740 manuscripts were extracted by using cardiovascular and oncological terms, respectively, and only 926 manuscripts were extracted by using both terms (Figure 1A), which indicates that the outcomes of miRNA studies might not be efficiently shared between the different disciplines. To bridge the gap between miRNA studies in cardiology and oncology, representative cardio-miRs upregulated in human heart failure were selected based on database screening. The targets and functions of these miR-NAs in cardiovascular diseases and cancers are comprehensively reviewed, and then circulating miRNA-based diagnostics for non-cancerous and cancerous diseases are discussed with a focus on personal diversity related to genetic and environmental factors.

REPRESENTATIVE CARDIO-miRs UPREGULATED IN HEART FAILURE
Heart failure is a progressive decline in cardiac functions that occurs at the end stage of cardiovascular diseases, such as ischemic heart disease, hypertension and diabetes (Hill and Olson, 2008;Shah and Mann, 2011;Zhou et al., 2013b). Myocardial infarction is caused by coronary artery occlusion, which leads to the death of cardiomyocytes in the infarcted region owing to insufficient oxygen supply. Ischemic stress occurs in surviving cardiomyocytes in the surrounding or peripheral area of an infarcted region, and then hypertrophic growth of myocardiocytes and interstitial fibrosis occur in the non-infarcted region of the heart. By contrast, persistent pressure overload causes cardiac wall thickening of the left ventricle and hypertrophic growth of cardiomyocytes. Cardiac hypertrophy leads to maladaptive remodeling of the left ventricle and eventually results in patient death owing to fatal arrhythmia and/or heart failure.
Forty-seven reports were recovered by initial screening of the literature in the PubMed and Web of Science (WoS) databases by using "heart failure," "miRNA or miRNAs," and "microarray." Then, four reports on microarray analyses (van Rooij et al., 2006;Matkovich et al., 2009;Naga Prasad et al., 2009;Zhu et al., 2013) were selected by critical reading (Figure 1B). Based on the criterion "miRNA that is upregulated in at least two reports on microarray analyses," miR-24, miR-125b, miR-195, and miR-214 were selected as candidate representative cardio-miRs that are upregulated in human heart failure ( Figure 1C). Because data obtained by using microarray analyses are not always correct, upregulation of miR-24, miR-125b, miR-195, and miR-214 in human heart failure were then validated by using a deep sequencing report on miRNA profiles in human heart failure (Leptidis et al., 2013). Based on the exploration and validation processes, miR-24, miR-125b, miR-195, and miR-214 were designated the representative cardio-miRs upregulated in human heart failure ( Figure 1B).

Involvement of miR-24 in cardiovascular diseases
miR-24 is upregulated in ischemic heart endothelial cells as a result of hypoxia-induced HIF-dependent transcription, but it is then transiently downregulated in adjacent surviving regions of acute myocardial infarction owing to the recovery of blood supply (Fiedler et al., 2011;Qian et al., 2011;Camps et al., 2014). miR-24 promotes cardiomyocyte survival through repression of pro-apoptotic Bim (Qian et al., 2011) and reduces cardiac fibrosis through repression of Furin protease that controls the activation of latent TGFβ . On the other hand, miR-24 inhibits the survival, migration, proliferation and tube formation of endothelial cells (angiogenesis) through repression of eNOS and actin cytoskeleton regulators, such as DIAPH1, LIMK2, and PAK4 (Fiedler et al., 2011;Meloni et al., 2013;Zhou et al., 2013a). miR-24 is upregulated in the chronic phase after myocardial infarction and promotes hypertrophic growth of cardiomyocytes in mouse model experiments and disturbs cardiac contraction through repression of JPH2 that is involved in the excitation-contraction coupling process of the heart (van Rooij et al., 2006;Xu et al., 2012b). Because miR-24 protects cardiomyocytes themselves and reduces cardiac fibrosis but inhibits angiogenesis and deteriorates heart failure, miR-24 is a multifunctional cardio-miR that plays good and bad roles in heart failure ( Figure 3A).

Involvement of miR-125b in cardiovascular diseases
miR-125b and LIN28A are human homologs of Caenorhabditis elegans (C. elegans) lin-4 and lin-28, respectively. C. elegans lin-4 is involved in the repression of lin-28 to orchestrate morphogenesis during larval stage, whereas human miR-125b is involved in the repression of LIN28A during the differentiation of embryonic stem cells (ESCs) into myocardial precursors and cardiomyocytes (Wu and Belasco, 2005;Wong et al., 2012). miR-125b is physiologically expressed in perivascular stromal cells rather than cardiomyocytes of the developing mouse heart (Schneider et al., 2011) and in cardiac valves rather than myocardium of the adult rat heart (Vacchi-Suzzi et al., 2013). miR125b is upregulated in mouse cardiac endothelial cells during endothelial-to-mesenchymal transition (EndMT) induced by TGF-ß (Ghosh et al., 2012). In addition, mir-125b is upregulated in early-stage cardiac hypertrophy after aortic banding (Busk and Cirera, 2010) and also in late-stage cardiac hypertrophy and heart failure (van Rooij et al., 2006). Ectopic miR-125b expression by using adenovirus vector does not elicit cardiomyocyte hypertrophy in vitro (van Rooij et al., 2006), whereas ectopic miR-125b expression by using lentivirus reduces myocardial infarct size and preserves cardiac functions in a mouse experimental model of acute myocardial infarction (Wang et al., 2014b). miR-125b is a good cardio-miR that protects the heart from ischemia/reperfusion injury ( Figure 3B).
miR-125b also functions as an oncogenic or tumor suppressor miRNA in a context-dependent manner ( Figure 3B).

Involvement of miR-195 in cardiovascular diseases
During early post-natal development of mice, miR-195 is upregulated in cardiac ventricles and induces cell-cycle arrest in cardiomyocytes through repression of cell cycle regulators, such as Cdc2a, Chek1, Birc5, Nusap1, and Spag5 (Porrello et al., 2011). Overexpression of miR-195 in the developing heart of transgenic mice by using the β-myosin heavy chain (MHC) promoter gives rise to perinatal cardiomyopathy in one line and ventricular hypoplasia and ventricular septal defects in another line (Porrello et al., 2011). Overexpression of miR-195 in primary neonatal rat cardiomyocytes by using adenoviral vector leads to hypertrophic growth and sarcomeric assembly, and overexpression of miR-195 in the heart of post-natal transgemic mice by using the α-MHC promoter gives rise to cardiac hypertrophy and dilated cardiomyopathy (van Rooij et al., 2006). In transgenic mice with the α-MHC mutation R403Q, miR-195 upregulation and subsequent repression of Cab39 in the heart leads to hypertrophic cardiomyopathy owing to inhibition of Lkb1/Strad/Cab39-dependent AMPK signaling . Together these facts indicate that miR-195 is a bad cardio-miR that elicits hypertrophic cardiomyopathy, dilated cardiomyopathy and heart failure ( Figure 3C).

Involvement of miR-214 in cardiovascular diseases
miR-214 is upregulated as a result of cardiac ischemia and heart failure. In a mouse model of ischemic cardiac injury induced by permanent ligation of the left anterior descending coronary artery, miR-214 prevents cardiomyocyte death owing to Ca 2+ overload, subsequent cardiac insufficiency and cardiac fibrosis through repression of Slc8a1 (Ncx1, sodium/calcium exchanger), which is the primary Ca 2+ outflow pump in cardiomyocytes (Aurora et al., 2012). miR-214 protects primary neonatal rat cardiomyocytes from apoptosis induced by ischemiareperfusion injury and represses Bim, Camk2d (Calmodulin kinase II delta) and Slc8a1 (Aurora et al., 2012). miR-214 also protects primary neonatal rat cardiomyocytes from apoptosis induced by H 2 O 2 through PTEN repression (Lv et al., 2014). Overexpression of miR-214 in transgenic mice under control of the α-MHC promoter does not induce a deteriorating cardiac phenotype; however, adenovirus-mediated pri-miR-214 delivery and lentivirus-mediated miR-214 delivery induce hypertrophic growth of primary neonatal rat cardiomyocytes in part through Ezh2 repression (van Rooij et al., 2006;Yang et al., 2013). miR-214 is a bi-functional cardio-miR that plays good and bad roles ( Figure 3D).

REGULATORY SIGNALING NETWORKS AND miRNA-BASED THERAPEUTICS
Regulatory signaling networks are defined as mutual interactions or cross-talks of receptor tyrosine kinase (RTK), G proteincoupled receptor (GPCR) and other receptor signaling cascades (Katoh, 2013a), which are involved in orchestration of fetal development and post-natal homeostasis as well as pathogenesis of non-cancerous and cancerous diseases. WNT, FGF Hedgehog, Notch, TGF-ß, BMP, Nodal, and Activin signaling cascades are major components of the regulatory signaling networks (Bailey et al., 2007;Katoh, 2007;Jayasena et al., 2008;Boulter et al., 2012;Nowell and Radtke, 2013;Coleman et al., 2014).

CIRCULATING miR-24, miR-125b, miR-195, AND miR-214
miRNAs function within the cell where they were produced as well as in other cells that receive miRNAs secreted or released from the cell of their origin (Valadi et al., 2007;Skog et al., 2008). Extracellular miRNAs are detected in various types of body fluids, such as blood, tears, saliva, urine, vitreous humor, cerebro-spinal fluid, pleural fluid, peritoneal fluid, seminal fluid, breast milk, and amniotic fluid (Mitchell et al., 2008;Weber et al., 2010;Ragusa et al., 2013). Extracellular miRNAs are classified into miRNAs in the blood (circulating miRNAs) and those in other body fluids. Because circulating miRNAs within exosomes (Taylor and Gercel-Taylor, 2008), microvesicles (Hunter et al., 2008) and high-density lipoprotein (Vickers et al., 2011) or those conjugated with AGO2 protein (Arroyo et al., 2011) are stable, circulating miRNAs are going to be utilized as diagnostics and prognostic biomarkers ( Table 3).
These facts clearly indicate that circulating miRNAs reported as cancer biomarkers are also dysregulated in non-cancerous diseases, and that miRNAs reported as biomarkers of non-cancerous diseases are also dysregulated in cancers (Table 3).

miRNA REGULATION BY GENETIC AND ENVIRONMENTAL FACTORS
Genetic factors are associated with individual traits and disease susceptibility (Lichtenstein et al., 2000;Zimmet et al., 2001;Milne et al., 2009). Single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) are major germ-line variations. The SNP rs1434536 is located in the miR-125b-binding site within the 3 -untranslated region (UTR) of BMPR1B. The C and T alleles of the rs1434536 SNP are sensitive and resistant to BMPR1B repression by miR-125b, respectively (Saetrom et al., 2009). The homozygous T genotype of rs1434536 is associated with increased risk of breast cancer (Saetrom et al., 2009) and decreased risk of endometriosis (Chang et al., 2013). Copy number loss of the miR-195 locus occurs in autism patients (Vaishnavi et al., 2013). Copy number gain of the miR-125b-2 locus occurs in Down syndrome patients as a result of trisomy 21, which leads to elevated circulating miR-125b in pregnant women with a Down syndrome fetus (Kotlabova et al., 2013) and causes acute megakaryocytic leukemia in Down syndrome patients (Klusmann et al., 2010). Genetic factors directly affect expression profiles and functions of miRNAs (Figure 5, upper left).
Environmental factors are also associated with disease susceptibility (Lichtenstein et al., 2000;Zimmet et al., 2001). Life style (food/beverage intake, tobacco smoking, air toxin, irradiation, etc.) and chronic infection (papilloma virus, hepatitis virus, Helicobater pylori, etc.) are environmental factors affecting individuals. Human miR-125b is downregulated in the bronchial epithelium of current smokers compared with never smokers (Schembri et al., 2009), and rat miR-125b is downregulated in the lungs of rats that were exposed to environmental smoke for 28 days (Izzotti et al., 2009). The expression profile of miRNAs in airway epithelial cells is altered by air toxins, such as diesel exhaust EZH2 and TET2 are epigenetic regulators that are involved in inactivation and activation of genes through repressive histone marking and CpG-island de-methylation, respectively. miRNA expression is downregulated by epigenetic silencing, while epigenetic regulators are repressed by multiple miRNAs. miRNAs and epigenetics are in the relationship of mutual regulation (lower part). Genetic and environmental factors regulate circulation miRNA profiles directly as well as indirectly through genetic and epigenetic alterations.
particles and formaldehyde (Jardim et al., 2009;Rager et al., 2011), whereas that in breast cancer cells is altered by endocrine disruptors, such as o,p'-dichlorodiphenyltrichloroethane (DDT), bisphenol A (BPA), fenhexamid and fludioxonil (Tilghman et al., 2012;Teng et al., 2013). The circulating miRNA landscape is altered by total-body γ-irradiation (Jacob et al., 2013) and by uptake of dietary polyphenols, such as quercetin, hesperidin, naringenin, anthocyanin, catechin, proanthocyanin, caffeic acid, ferulic acid and curcumin (Milenkovic et al., 2012), in model animal experiments. miRNA expression profile is altered by chronic infection with human papilloma virus, hepatitis virus and Helicobacter pylori, which are involved in pathogenesis of cervical cancer (Wang et al., 2008b), HCC (Ladeiro et al., 2008;Arzumanyan et al., 2013), and gastric cancer , respectively. Environmental factors directly alter circulating or tissue levels of miRNAs (Figure 5, upper right). Genetic alterations, such as gene amplification, deletion, translocation, point mutation or single nucleotide variation (SNV), occur in tumor cells during multi-stage carcinogenesis owing to mutual interactions of genetic and environmental factors (Lichtenstein et al., 2000;Katoh et al., 2013c;Katoh and Nakagama, 2014). SNVs in diffuse large B-cell lymphomas that disrupt the miR-125b-binding site within the 3 -UTR of TP53 are associated with better prognosis of patients owing to derepression of a tumor suppressor TP53 . Effects of gene amplification, deletion and translocation on expression profiles of miR-24, miR-125b, miR-195, and miR-214 have been described above ( Table 2). Genetic alterations play a key role for the regulation of miRNA profiles in somatic cells (Figure 5, upper middle).
Genetic and environmental factors dynamically alter expression profiles of miRNAs in individuals and also indirectly alter miRNA profiles through genetic and epigenetic alterations in patients with non-cancerous diseases and cancers (Figure 5).

CIRCULATING miRNA-BASED DIAGNOSTICS
Circulating miR-195 is upregulated in colorectal adenoma (Kanaan et al., 2013); however, miR-195 in colorectal adenoma FIGURE 6 | Circulating miRNA association study (CMAS). Dysregulation of circulating miRNAs occur in a variety of human disease. Circulating miRNA profiles in several thousands of cases each for non-cancerous common diseases (blue box) and major cancers (red box) should be investigated for the establishment of miRNA-based diagnostic platform.
tissues is repressed owing to epigenetic silencing and deletion (Menigatti et al., 2013). Circulating miR-125b, miR-195, and miR-214 are upregulated in breast cancer patients ( Table 2), whereas these miRNAs in breast cancer tissues are downregulated owing to epigenetic silencing or deletion ( Table 2). These facts clearly indicate that circulating miRNAs in cancer patients are not always derived from tumor tissues.
Because circulating miRNA profiles are dynamically regulated by genetic and environmental factors (Figure 5), circulating miRNA profiles of cancer patients reflect co-existing non-cancerous diseases or individual whole-body conditions. Therefore, circulating miRNA association studies (CMASs) within several thousands of cases each for common noncancerous diseases as well as major cancers (Figure 6) should be carried out to establish a reliable and robust platform of miRNA-based diagnostics.

CONCLUSION
Cardio-miRs and onco-miRs bear some similarities in functions and circulation profiles. miRNAs modulate the regulatory signaling networks that are involved in orchestration of embryogenesis and homeostasis as well as pathogenesis of human diseases. Circulating miRNA profiles within several thousands of cases each for non-cancerous and cancerous diseases are necessary for the establishment of miRNA-based diagnostics.