Identification of a New Target of miR-16, Vacuolar Protein Sorting 4a

Rationale The rationale was to utilize a bioinformatics approach to identify miRNA binding sites in genes with single nucleotide mutations (SNPs) to discover pathways in heart failure (HF). Objective The objective was to focus on the genes containing miRNA binding sites with miRNAs that were significantly altered in end-stage HF and in response to a left ventricular assist device (LVAD). Methods and Results BEDTools v2.14.3 was used to discriminate SNPs within predicted 3′UTR miRNA binding sites. A member of the miR-15/107 family, miR-16, was decreased in the circulation of end-stage HF patients and increased in response to a LVAD (p<0.001). MiR-16 decreased Vacuolar Protein Sorting 4a (VPS4a) expression in HEK 293T cells (p<0.01). The SNP rs16958754 was identified in the miR-15/107 family binding site of VPS4a which abolished direct binding of miR-16 to the 3′UTR of VPS4a (p<0.05). VPS4a was increased in the circulation of end-stage HF patients (p<0.001), and led to a decrease in the number of HEK 293T cells in vitro (p<0.001). Conclusions We provide evidence that miR-16 decreases in the circulation of end-stage HF patients and increases with a LVAD. Modeling studies suggest that miR-16 binds to and decreases expression of VPS4a. Overexpression of VPS4a decreases cell number. Together, these experiments suggest that miR-16 and VPS4a expression are altered in end-stage HF and in response to unloading with a LVAD. This signaling pathway may lead to reduced circulating cell number in HF.


Introduction
MiRNAs regulate gene function in heart development, physiology and pathology. The miRNA-mRNA interaction is affected by the presence of single nucleotide polymorphisms (SNPs). A SNP within or near the miRNA binding sites of targets has been shown to influence the susceptibility to disease [1,2]. In this study we identified new miRNA/RNA targets using a novel approach of utilizing conserved SNPs in miRNA binding regions as a roadmap. Determining how short RNAs (miRNA and siRNA) impact mRNA target expression within single cells and how this process influences disease initiation and progression is still not known. Recent work has suggested that within each cell, 85% of genes contain fewer than 100 copies [3]. Mullokandov et al provide evidence that only the most abundant miRNA regulate target gene expression [4]. The turnover rate of miRNA has been shown to alter gene expression [5], as well as the secondary structure of the target gene [6].
Several publications have cited circulating expression of miRNA in end-stage HF; however it is challenging to find how the levels of these miRNA compare to others in the circulation [7][8][9][10][11]. These studies identified increases in miR-423-5p and miR-499, and a decrease in miR-126 [7][8][9][10][11]. MiR-16 is one of the highest expressed mRNAs in the circulation. Expression of miR-16 is widely known to be high in all circulating cells [12]. Reticulocytes (immature red blood cells) and platelets exhibit the highest expression of miR-16 [12]. The role of miR-15/107 family members, including miR-16 in peripheral blood in the context of HF has not been defined. MiR- 15 -16, -195, -497, -103, -107, -503, share a common sequence ''AGCAGC'' starting at the first or second nucleotide from the 59 mature end of the miRNA [13]. This evolutionary conserved family of miRNAs targets a disproportionately high number of cell cycle regulatory genes [14][15][16][17][18]. A predicted target, Vacuolar Protein Sorting 4a, VPS4a, was identified with a SNP rs16958754 in the miR-15/107 family binding site. VPS4a is an ATPase and is one of many vacuolar protein sorting associated proteins involved in diverse cellular processes including multivesicular body formation, cytokinesis, and endosomal trafficking [19].
In this study, we identify miR-16 as the specific member of the miR-15/107 family that decreases in peripheral blood of patients with end stage HF and increases significantly one week post ventricular assist device (LVAD) therapy. The novel target of miR-16, VPS4a that we identified and validated in vitro, increases in peripheral blood of end stage HF patients and decreases one week following LVAD therapy. MiR-16 has been identified in CD45 and CD14 positive cells [12]. Overexpression of VPS4a significantly inhibited total cell number, suggesting that the miR-16-VPS4a pathway inhibits proliferation of CD45 and CD14 positive cells in end stage HF.

Methods
An expanded methods section (Text S1) is available as Supporting Information.

Ethics statements
All procedures followed were in accordance with the ethical standards of the responsible committees on human experimentation. Written consents were obtained from eligible patients for being included in this study. Samples were collected from consenting participants and the study was conducted under the Institutional Review Board approval from the University of Minnesota, USA the Royal Brompton and Harefield Cardiovascular Biomedical Research Unit, UK and Stanford University. All samples used in the study and the original repository are listed in Table S5.

Heart samples
Heart samples from patients with end-stage HF were collected at the time of LVAD implant (HF, n = 10) at the University of Minnesota. Non-failing control heart samples (Control, n = 9, 4565, 57% male) were obtained from Dr. Euan Ashley, Stanford University.

Peripheral blood samples
Blood samples from patients with end-stage HF (n = 9) were collected prior to LVAD implant and 7 days post-implant (n = 9) as described [20] (Table 1). Blood samples from healthy gender and age matched subjects were labeled as controls (n = 6, average age 5464, 60% male, 83% Caucasian).

DNA samples, genotyping and analysis
DNA samples were obtained from individuals (n = 855) from the Royal Brompton and Harefield NHS Trust Tissue Typing Service, UK, referred for cardiac transplantation due to dilated cardiomyopathy (DCM, n = 234) or ischemic cardiomyopathy (ICM, n = 621), and from individuals at the University of Minnesota (n = 152) referred for heart transplant or placement of a LVAD (DCM, n = 67 and ICM, n = 76) all according to institutional approved protocols. Primers as listed in the Table S4 were used to genotype samples for SNPs in miRNA binding sites on the commercially available high-throughput genotyping Sequenom MassARRAY platform (San Diego, CA, USA) as previously described [21]. SNPs were compared to that in a general population (Caucasian samples from 1000 Genome project, Phase 1 data, (http://www.1000genomes.org/). We identified 853 unique SNPs in 914 unique target sites in 763 unique 39UTRs in dbSNP. From ESP5500 we identified 343 unique SNPs that were found in 348 unique target sites in 311 unique 39UTRs (starting from an initial size of 14,024,295 SNPs, and 1,837,369 SNPs in the ESP5500 database). For details see Text S1 and Table  S4.

RNA isolation and Real Time Quantitative PCR (RTQPCR)
Total RNA was extracted with Trizol according to the manufacturer's directions (Invitrogen). For miRNA, cDNA were synthesized by using the Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems). For mRNA expression cDNA was synthesized by utilizing the SuperScript III kit (Invitrogen) [22]. Data is expressed relative to control and was calculated by the 2 ' -deltadeltaCT method as described [22]. Taqman primers used in this study are listed in the Table S4.

Dual Luciferase Reporter Assay
HEK 293T cells were co-transfected with either WT or mutated (harboring rs16958754) VPS4a 39UTR reporter clones for miRNA target validation (Origene) and Renilla luciferase construct (Promega) with miR-16, -103, -107 or a scrambled control mimic according to the manufacturer's protocol (Life Technologies). The 39UTR of VPS4a was cloned downstream of the luciferase gene. An Internal ribosome entry site (IRES) is located upstream of luciferase. As the miR/RISC complex binds to the target, luciferase activity is reduced. If there is no interaction between the miR and the target, luciferase activity remains high. Luciferase activity was assessed using the Dual Luciferase Reporter Assay System (Promega) [23].

Western Analysis
For details see Text S1.

Cell number and Apoptosis
HEK 293T cells were plated and transfected with VPS4a cDNA plasmid (Origene) or an empty vector (Origene) using Lipofectamine 2000 according to the manufacturer's instruction (Life Technologies). Increase in cells number was assessed every 24 h using a Hemocytometer. Apoptosis was assessed 48 h post transfection by staining with Hoechst 33342 for nuclear morphology, and the percentage of apoptotic nuclei was quantified as previously described [24].

Statistics
A Student's T-test was used for comparison of two groups and a One Way ANOVA with a post-hoc Tukey analysis was used for VPS4a Is Regulated by miR-16 PLOS ONE | www.plosone.org Table 1. Characteristics of patients with end-stage HF. Blood samples were obtained prior to LVAD implantation (n = 9) and 7 days post implant (n = 9). P a t i e n t I D

Results
We identified 1175 SNPs in miRNA binding sites within the 39 UTR of genes (Table S1). This supplemental table includes targets harboring the same SNP within the binding sites of multiple miRNA families. 137 genes contained a SNP in the 39 UTR that was within the binding site of more than one miRNA family (Table S1).
24 SNPs were identified in binding sites specific for the miR-15 family. 11 SNPs were identified in binding sites specific for the miR-107 family. One SNP was shared between the miR-15 and the miR-107 family, rs16958754 [C/T] in VPS4a (Fig. 1). miR-15a, -16, -195 and -103 were decreased in peripheral blood samples of individuals with end-stage HF compared to controls (gender, age, and ethnicity matched) (p,0.02, Fig. 2A)    MiR-16 in the peripheral blood was the only miRNA to increase 7 days post LVAD implantation (p,0.001, Fig. 2A). Heart tissue from end-stage HF patients (n = 10) showed no change in expression of miR-15a, -16, -195 and -103 compared to controls (n = 9, Fig. 2B).
VPS4a (the gene containing rs16958754 in the miR-15/107 binding site) was increased in peripheral blood from end-stage HF patients and decreased 7 days post-surgery as compared to controls (p = 0.0001) (Fig. 3A). VPS4a expression in heart tissue from HF patients was not different from non-failing controls (Fig. 3B).
The two separate cohorts of end-stage HF samples were genotyped for a subset of the SNPs. The minor allele frequency (MAF) of these SNPs in the two HF populations did not differ significantly from the MAF published in dbSNP or 1000 genomes (Table S2). No SNP showed significant frequency difference between ethnic groups. Using data from the 1000 genomes project (http://www.1000genomes.org/), we were able to map 63 miRNA SNPs by their physical position on the genome. MAFs of these SNPs in Caucasians were compared between our samples and the 1000 genomes samples, and no significant difference was observed. Of interest, we observed several individuals carrying multiple alleles at multiple loci at low frequencies. Specifically, four individuals carry 4 rare alleles (at 4 loci) with frequency ,1%, and nine individuals carry 3 rare alleles with frequency ,1%. Assuming independence among the miRNA loci, we randomly permuted the rare alleles in our samples to estimate the expected number of individuals carrying multiple rare alleles ( Table 2). The excess number of individuals than expected may suggest correlation or interaction between the miRNA loci surveyed in our study.

Discussion
A major finding of this study is that 2-3 miRNA families may bind to a site in the 39 UTR that contains a single SNP. One of these SNPs, rs16958754, in the gene, VPS4a, was identified in the binding region of both miR-15 and miR-107 families. These families share a common sequence, ''AGCAGC''. miR-16, a member of the miR-15 family, is one of the top 10 highest expressed mi-RNAs in peripheral blood cells [12]. Expression of miR-16 in the blood was down-regulated in end-stage HF and upregulated 7 days post unloading with a LVAD. In contrast, the target of miR-16, VPS4a, was up-regulated with HF, and downregulated post LVAD in the blood (Fig. 3A). Studies in vitro validated that miR-16 decreased mRNA level of VPS4a. The presence of the SNP, rs26958754 abolished this effect. Although the presence of the SNP, rs16958754, interfered with miR-16 interaction with VPS4a, the MAF of this SNP in our two heart failure cohorts was rare (0.7 and 2.2%). The MAF of this SNP in normal controls in the 1000 genome project was 0. Taken together, these experiments suggest that miR-16 interacts with VPS4a and that circulating expression of miR-16 is downregulated with HF while expression of its target, VPS4a, is increased. In response to LVAD therapy, circulating miR-16 is increased along with a concomitant decrease in VPS4a. In the rare case when a subject carries the SNP, rs16958754, it is possible that VPS4a levels have been high prior to HF given that miR-16 was elevated and not binding to the 39UTR of VPS4a. It is speculated that VPS4a regulation through miR-16 would remain high during HF, given miR-16 is decreased and that miR-16 does not bind efficiently to VPS4a in patients with the SNP. Finally, it is possible that LVAD therapy would not decrease VPS4a through a miR-16 mediated mechanism in these subjects, given that miR-16 would increase with the LVAD yet not interact with its target VPS4a due to the SNP, rs16958754.
To define the cellular role of increased VPS4a in heart failure, we overexpressed VPS4a in HEK 293T cells and showed a significant decrease in cell number. This data agrees with the majority of studies showing that a decrease in miR-16 decreases cell number [25][26][27]. However not all studies show that miR-16 decreases cell number, which one might expect given miR-16 has multiple downstream targets [28]. VPS4a is thought to be part of an evolutionary ancient membrane remodeling system [19]. VPS4a is regulated by miRNAs 34a, 34c-5p, 449a, 449b, 574-3p, and -515-3p. To our knowledge, these miRNAs have not been reported to be altered in response to HF or LVAD therapy in humans. Furthermore, unlike miR-16, these miRNAs are not expressed at a high level in peripheral blood cells [12]. Expressions of many of the top miRNAs in peripheral blood are increased by 3-4 folds in platelets and reticulocytes including miR-16.
SNPs within miRNA binding sites are currently being used as prognostic and predictive cancer biomarkers [29]. The list of SNPs provided in this study may lead to future work in this direction in the area of cardiovascular disease as well as other diseases outside the realm of cardiovascular disease. The potential to utilize these SNPs and their targets as biomarkers for response to treatment are also future possibilities. We identified many targets with SNPs within a binding site that was recognized by multiple miRNAs. The potential for these SNPs and gene targets to be prognostic markers for blood diseases or immune related diseases in which T and B cells are critical looks to be very promising. This is particularly interesting given that many of the patients have 2 SNPs that share a common miRNA that is regulated in the context of cardiovascular disease including miR-129. The complete list of the sample reference numbers and SNPs, miRNAs, and gene targets with more than one SNP is given in the Table S3. In sum, this study shows that SNPs in miRNA binding sites, and/or levels of miRNA expression may impact the progression of heart failure and/or the response to treatment such as a LVAD.
The potential limitations of this study are that the HF cohorts we used to genotype were relatively small. We did genotype two cohorts (one from Minnesota and the other from London), both with end-stage HF. We have provided all of the SNPs identified in the miRNA binding sites for others to utilize. Secondly, the cell type we utilized for the modeling experiments were HEK 293T cells, an epithelial cell line derived from human embryonic kidney. Although experiments in primary cells may have been insightful, the goal was to test if VPS4a was indeed a downstream target of miR-16 and if the SNP interfered with the binding. For these experiments the HEK 293T cells provided clean, interpretable data.
In conclusion, the present study identified and validated VPS4a as a novel target of miR-16. The SNP rs16958754 [C/T] interfered with the binding of miR-16 to VPS4a. Loss of miR-16 in patients with end stage HF and the concomitant rise in VPS4a may lead to a significant decrease in circulating platelets and/or reticulocytes with end stage disease. In response to treatment with a LVAD, miR-16 increases and VPS4a decreases. This suggests a potential role for the miR-16-VPS4a signaling pathway in erythropoiesis.   The complete list of sample ID, SNPs, miRNAs, and gene targets with more than one SNP is given in the Table S3