A polymorphism rs3746444 within the pre‐miR‐499 alters the maturation of miR‐499‐5p and its antiapoptotic function

Abstract microRNAs (miRNAs) are non‐coding RNAs that function as post‐transcriptional regulators of cardiac development and cardiovascular diseases. Single nucleotide polymorphisms (SNPs) in miRNA genes are a novel class of genetic variations in the human genome that confer the risk of cardiovascular diseases. Here, we identified a polymorphism A→G (rs3746444) in miR‐499 precursor (pre‐miR‐499) that affects the maturation of miR‐499‐5p and alters its antiapoptotic function by converting stable A‐U base pair to wobble G‐U base pair in pre‐miR‐499 secondary structure. Furthermore, our results showed that the concentrations of plasma miR‐499‐5p could be correlated with myocardial infarction (MI) and heart failure (HF) patients in comparison with control subjects and polymorphism rs3746444 in miR‐499 could influence its abundance in plasma. Finally, our results also showed that the variant of polymorphism in miR‐499 influenced the severity of the myocardial infarction significantly. This is the first report to highlight the biological significance of this polymorphism on the maturation of miR‐499‐5p and its antiapoptotic role during MI. These findings may pave a way to better understand the individual variability based on miRNA SNPs in heart diseases and may contribute to better treatment for disease severity on a personalized level.

may affect different signalling pathways by altering the expression levels of downstream genes. It has been shown that SNPs in miRNA genes of the individuals are associated with the susceptibility to many diseases, including cancer and cardiovascular disease. [10][11][12][13][14] Previous studies reported that miR-146a rs2910164, miR-149 rs71428439, miR-196a2 rs11614913 and miR-499 rs3746444 might be related to the risk of heart diseases. 13,[15][16][17] Studies have shown that the pre-mir-146a and pre-mir-149 polymorphisms alter their miRNA expression levels. 15,18 Previous reports showed that naturally occurring miR-499-5p mutation outside the critical seed sequence (miR-499-5p c17) modifies mRNA targeting and end-organ function of miR-499-5p. 19 However, it is not known whether the mechanism of genetic variants located in pre-miR-499 associated with the susceptibility to heart disease and whether pre-mir-499 rs3746444 affects the maturation and expression of miR-499-5p. miR-499-5p is a cardiac-abundant miRNA under physiological conditions. 20,21 It is regulated and functioned in heart development. 21 Several studies revealed that miR-499-5p levels were down-regulated under pathological conditions. 22 Furthermore, miR-499-5p plays a cardioprotective role by inhibiting the apoptosis in cardiomyocytes and regulates the mitochondrial fission machinery. 22,23 miR-499-5p targets several pro-apoptotic regulators such as programmed cell death protein 4 (Pdcd4), SOX6,calcineurin catalytic subunit α-form (CnAα) and βform (CnAβ). [22][23][24] In vivo studies suggested that miR-499-5p can inhibit myocardial infarction and cardiac remodelling upon ischaemicreperfusion, 22 and its modulation may provide a therapeutic approach for treating myocardial infarction. Whether genetic variant in pre-miR-499 affecting its antiapoptotic function remains to be revealed.
Dysregulated miRNA expression patterns have been associated with various cardiac pathologies. 25 Therefore, it may be considered that altered miRNA expression can be an indicator of heart damage that happened in pathological conditions. In particular, since the discovery of circulating miRNAs in the bloodstream, many studies are being performed to explore the association of miRNAs, expression levels and development of any particular disease to use miRNAs as diagnostic markers. 26,27 The previous reports have associated the elevated levels of miR-499-5p in plasma of subjects with acute myocardial infarction (MI) and below the detection levels in congestive HF. Therefore, the plasma levels of miR-499-5p may be helpful for distinguishing acute MI from cardiopathy in the cardiac patients. 28 However, the field is still in its infancy and the mechanistic details of the differential expressions of miRNAs and its consequences are to be elucidated. Furthermore, whether SNPs located in these miRNAs may affect the sensitivity of plasma miRNAs, as diagnostic markers, is still uncovered.
Taking all these aspects into consideration, this study was aimed at exploring the mechanism that associates SNP rs3746444 of pre-miR-499 with the susceptibility of heart disease. Here, we report that the G allele miR-499 precursor displays the decreased production of mature miR-499-5p compared with the A allele, which further affect the antiapoptotic function of miR-499. We also found that plasma miR-499-5p can be used as diagnostic markers for MI; however, its genetic variants influence its levels in the plasma of patients. Therefore, the identification of the functional genetic variants in the miRNAs, which are associated with heart diseases, may improve the precision diagnostics of cardiac patients and improve our knowledge of their pathophysiology.

| Study subjects
All study subjects were recruited from Affiliated Hospital of Qingdao University, Qingdao, China. Approval for this study was obtained from the medical ethics committee of the Affiliated Hospital of Qingdao University. This study included 29 patients with myocardial infarction (MI group), 30 patients with chronic heart failure (HF group) and 14 asymptomatic controls (control group). Informed consent was obtained from all study subjects. Inclusion criteria for the HF group were chronic stable HF diagnosed according to Framingham standards, New York Heart Association Stage III-IV and plasma prohormone of brain natriuretic protein (pro-BNP) content ≥1000 ng/L. Inclusion criteria for the MI were based on the following criteria: (a) typical chest pain lasting longer than 30 minutes; (b) characteristic electrocardiographic patterns of myocardial infarction; (c) elevation of cardiac enzymes (creatine kinase and lactate dehydrogenase) and troponin I or T in blood. The control individuals were randomly selected from individuals without history of cardiovascular events and signs of dysfunction of the cardiovascular system by physical examination. These control subjects were frequency-matched to the cases on age and sex.
All subjects were genetically unrelated ethnic Han Chinese from Qingdao city and surrounding regions.

| RNA extraction
Blood samples were obtained from antecubital vein of each subject.
After rapid centrifugation, plasma samples were transferred to RNase/ DNase-free tubes and stored at −80°C. Total RNA was isolated from 500 μL of plasma with TRIzol reagent (Invitrogen, Grand Island, NY).
The plasma levels of miR-499 were measured using a qRT-PCR assay.

| Construction of pri-miR-499 expression vectors
To create the allelic A and G pre-miR-499 expression vectors separately, the 600-bp DNA fragments encompassing the miR-499 precursor DING ET AL.
| 5419 sequence and its 5-and 3-flanking regions (281 and 197 bp, respectively) were amplified from human genomic DNA from subjects included in the study (determined to have the AA or GG genotype) and cloned into the XhoI and XbaI sites of the vector pcDNA3.1 (Invitrogen). The sequences of both vectors were confirmed by direct sequencing, and the only difference was in the site of SNP. The yielded vector with the AA genotype was designated as pri-miR-499-A, and the one with the GG genotype was named as pri-miR-499-G.

| Cell transfection with miRNA duplexes or miRNA inhibitors
The miR-499-5p duplexes were synthesized by GenePharma Co. Ltd

| Immunoblotting
Immunoblotting was performed as described previously. 31 Briefly, cells were lysed for 1 hour at 4°C in a lysis buffer containing a protease inhibitor cocktail. Protein samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed using corresponding primary antibodies. Then the horseradish peroxidase-conjugated secondary antibodies were used. Antigenantibody complexes were tested by enhanced chemiluminescence.
The antibodies to CnAα and CnAβ were purchased from Millipore.

| Statistical analysis
The results are expressed as means ± SD of at least three independent experiments. The differences among experimental groups were evaluated by one-way analysis of variance for comparison among three or more groups and the two-tailed t test applied for the comparison between two groups, respectively. P < 0.05 was considered statistically significant. For miRNAs with significant differences and associations, the ability to discriminate between the MI and control groups was characterized by the receiver operating characteristic (ROC) curve, and the area under the ROC curve (AUC) was estimated to assess the diagnostic accuracy of miRNAs. Statistical analyses were performed with SPSS 13.0. ROC curve was evaluated with GraphPad Prism 5.

| The polymorphism rs3746444 changes A-U pair to G-U pair in miR-499
The miR-499 gene is located within intron 20 of human cardiac βmyosin heavy chain 7B gene (MHC7b) in 7q11. 21 miR-499-5p and miR-499-3p are derived from the 5′ and 3′ arms of pre-miR-499 hairpin precursor, respectively. miR-499-5p is the guide strand of miR-499 gene and highly expressed in cardiac tissue under physiological conditions. 21 The polymorphism, rs3746444, is located at the 73rd position of pre-miR-499 (the 6th position of miR-499-3p).
Therefore, it is referred as miR-499 A73G SNP. Alignment of available pre-miR-499 sequences revealed that miR-499 SNP, rs3746444, is highly conserved in mammals ( Figure 1A). The nucleotide C73 is located in the stem of pre-miR-499 and forms an A-U base pair with nucleotide U49 ( Figure 1B). The A73G polymorphism changes the conserved A-U base pairs to G-U wobble pairs ( Figure 1B). We used mfold web server 32 to predict the minimum free energy for the secondary structure of pre-miR-499-A and pre-miR-499-G to detect the effect of this variant on the secondary structure of pre-miR-499. We did not observe a significant change in the minimum free energy by altering this nucleotide A to G ( Figure 1C). Although this polymorphism did not affect secondary structure of pre-miR-499, the alteration of stable A-U base pair to wobble G-U base pair could potentially affect miR-499 maturation, including primary miR-499 (pri-miR-499) processing by Drosha, pre-miR-499 processing by Dicer and/or miR-499 duplex stability.

| The polymorphism rs3746444 affects the maturation of miR-499-5p
To explore whether the presence of the polymorphism rs3746444 in pre-miR-499 affects production of miR-499-5p, miR-499-5p abun- quantified the abundance of mature miR-499-5p by qRT-PCR. In contrast, the A73G SNP markedly reduced the levels of mature miR-499-5p in HEK-293 cells ( Figure 2C). Furthermore, the expression levels of miR-499-5p were also altered between different alleles in H9c2 cardiomyocytes transfected with above plasmid. The abundance of mature miR-499-5p was reduced in the G allele plasmids transfected cells in comparison with A allele ( Figure 2D). miR-499-5p is the dominant mature miRNA product of miR-499 gene as shown in the microRNA database miRBase. Whether the polymorphism rs3746444 affects miR-499-3p level is unknown. We detected miR-499-3p level in cardiomyocytes. Comparing to miR-499-5p,the level of miR-499-3p in cardiomyocytes is rather low ( Figure 2E). Next, HEK-293 cells were transfected with pri-miR-499-A or pri-miR-499-G and miR-499-3p level was detected. However, miR-499-3p level was not significantly affected ( Figure 2F). It indicated that miR-499-3p is rarely expressed and not affected by this SNP. These data suggested that the A to G substitution in pre-miR-499 affects the maturation of miR-499-5p. We concluded that the miR-499 SNP reduces levels of miR-499-5p, without affecting the expression levels of pri-miR-499.

| The polymorphism rs 3746444 affects antiapoptotic function of miR-499-5p
In our previous study, we have reported that miR-499-5p could inhibit apoptosis and myocardial infarction. 22 Figure 3E). Therefore, these result suggested that the polymorphism rs 3746444 affects antiapoptotic function of miR-499-5p.

| The polymorphism rs3746444 reduces the suppression of miR-499-5p target
As the A73G SNP reduced the levels of miR-499-5p, we expected that the SNP might affect the target suppression of miR-499-5p. We reported previously that both isoforms of calcineurin catalytic subunit, the α-form (CnAα) and β-form (CnAβ) are the targets of miR-499-5p. 22 Therefore the effect of pri-miR-499-A and pri-miR-499-Gallelic variants on the translation of CnAα and CnAβ was tested by luciferase reporter assay. Cotransfection of CnAα 3′-UTR reporter plasmid with pri-miR-499-A plasmid resulted in significant decrease in the luciferase activity. However, the target suppression ability of the pri-miR-499-G allele was significantly weaker than the pri-miR-499-A allele by comparing their luciferase activities ( Figure 4A). Similarly, cotransfection of CnAβ 3′-UTR reporter plasmid with pri-miR-499-A plasmid resulted in a marked decrease in luciferase activity, but suppression of the pri-miR-499-G allele plasmid was again weaker for this target than miR-499-A-allelic plasmid ( Figure 4B).
The overexpression of miR-499-5p led to an obvious reduction in both of its targets, CnAα and CnAβ. However, the reduction in the expression levels of both targets was significantly stronger in the presence of miR-499-A allele than miR-499-G allele ( Figure 4C). This evidence suggested that the miR-499 SNP reduces the suppression of miR-499-5p target, which is consistent with decreased expression levels of miR-499-5p due to the presence of this polymorphism.

| Plasma miR-499-5p serves as a diagnostic marker for heart disease
The circulating miRNAs are being assessed as diagnostic and prognostic biomarkers because the altered levels of some miRNAs have been reported in human body fluids for different cardiac diseases. 33 Plasma miR-499-5p has also been suggested as a biomarker of acute MI and CHD previously. 28,34,35 We assessed the plasma concentrations of miR-499-5p in 29 individuals with acute MI, 30 individuals F I G U R E 3 The rs3746444 polymorphism affects antiapoptosis function of miR-499-5p. A, miR-499-5p levels were detected in H9c2 cells exposed to 100 μmol/L H 2 O 2 at the indicated time. *P < 0.05 vs control. B and C, G-allelic miR-499 precursor inhibited apoptosis at a lesser degree compared with the A-allelic one. H9c2 cells were transfected with the plasmid of pri-miR-499-A or pri-miR-499-G, respectively. After 24 hours, cells were exposed to 100 μmol/L H 2 O 2 for 24 hours. miR-499-5p levels (B) and apoptosis (C) were analysed. Representative photographs showed TUNEL-positive cell (C, left with HF, with a median score of 0.664 in the HF group, than the control subjects, with a median score of 0.941 ( Figure 5C).

| The allelic variants of polymorphism, rs3746444, affect the plasma levels of miR-499-5p and its clinical features in MI patients
The polymorphism rs3746444 significantly influencing the maturation of miR-499-5p motivated us to further study whether this SNP genotype affects the level of plasma miR-499-5p and clinical features for MI patients. The levels of miR-499-5p were statistically different between MI patients with AA genotype compared to those with AG or GG genotypes. As shown in Figure 6A, the plasma miR-499-5p levels of MI patients with AA alleles increased about 3.69-fold compared with normal population, which was significantly higher than the other two alleles AG (2.2 folds) and GG (1.43 folds). While in normal population, the level of plasma miR-499-5p was not affected by this SNP genotype. ( Figure 6B). However, the sample size was small, it need to be further verified. Therefore, it can be inferred that patients with AA genotype of miR-499 are most sensitive to MI diagnosis in comparison with AG and GG genotype.
We further compared the clinical feature of MI patients based on the miR-499 SNP genotype. It was observed that left ventricular ejection fraction (LVEF) in patients carrying the G allele in the genome was significantly lower than that of patients carrying the A allele ( Figure 6C). Taken together, these results suggested that the polymorphism rs3746444 could significantly influence the severity of heart disease and diagnostic sensitivity of MI when considering miR-499-5p as a biomarker for disease detection.

| DISCUSSION
The human genome is enriched with polymorphisms that result in different phenotypes. Accumulating evidence suggests that miRNAs F I G U R E 4 The rs3746444 polymorphism reduces target suppression by miR-499-5p. A and B, G-allelic pre-miR-499 causes a lesser inhibition of CnAα-3′UTR and CnAβ-3′UTR translation activity than the A-allelic one. HEK293 cells were transfected with the luciferase constructs of CnAα-3′UTR (A) or CnAβ-3′UTR (B), along with the plasmids for empty pcDNA3.1, pri-miR-499-A or pri-miR-499-G, respectively. Luciferase activity was measured after 36 hours. pGL3 served as a control. Data are expressed as the mean ± SD of 3 independent experiments. *P < 0.05. C, G-allelic pre-miR-499 suppresses the expression of CnAα and CnAβ at a lesser degree than the A-allelic one. H9c2 cells were transfected with the plasmids for empty pcDNA3.1, pri-miR-499-A or pir-miR-499-G, respectively. Twenty-four hours after infection, CnAα and CnAβ protein levels were detected by immunoblot. A representative blot of three independent experiments is shown play important regulatory roles in different diseases including cardiovascular disease. 25 The polymorphisms in miRNAs have been linked to susceptibility to cardiovascular diseases such as myocardial infarction and coronary heart disease. 13,16,36 The SNPs in miRNA genes may influence the processing and/or target selection of miRNAs. 15 In this study, we first identified that SNP rs3746444 located in miR-499 precursor can affect the expression levels and antiapoptotic function of miR-499-5p, which have been associated with MI and HF. We also observed that the individual's genotype of the SNP rs3746444 alters the diagnostic sensitivity of circulating miR-499-5p marker for the cardiovascular diseases.
Although the association between the SNPs in protein-coding genes and the risk of many kinds of diseases has been extensively investigated, there are only a few reports about the SNPs in miRNAs which are associated with risk of cardiovascular diseases. Previous studies have shown that several miRNA polymorphisms (miR-146a rs2910146, miR-149 rs71428439, miR-499 rs37464444, miR-208 rs11134527 and miR-149 rs71428439) contribute to the risk for the development of cardiovascular diseases including CHD, HF and MI. 13,15,18,36 The future studies are required to further explore the roles of SNPs in miRNA related to the risk of other cardiovascular diseases including cardiomyopathy.
As miR-499-5p is highly expressed in cardiac tissue and participates in the maintenance of cardiac survival and function, 21,25 it is reasonable to propose that polymorphism rs3746444 may contribute to the susceptibility of heart diseases. For the polymorphism rs3746444 (A→G) of hsa-mir-499, the few previous studies have shown that the variant genotype GG of this SNP was significantly associated with the increased risk of cancer 37 and dilated cardiomyopathy, 38 but most of the studies found no significant association with the increased risk of cancer. [39][40][41] It has been revealed that the GG and AG genotypes of SNP rs3746444 conferred increased risk for CHD 13 and ischaemic stroke, 42 respectively. Furthermore, the GG genotype increased the risk of MI in a Chinese population, 16 indicating that MI may share genetic risk factor with both CHD and ischaemic stroke. However, Xiong et al 18 found no association between SNP rs3746444 and CHD. One potential explanation for this phenomenon may be different genetic backgrounds among races.
The possible mechanism for such associations might be due to impaired maturation of miR-499-5p in GG homozygotes in comparison with AA homozygotes. The variation of a single base in the genes of miRNAs is able to influence maturation of miRNAs or alters their target sites, ultimately results in the altered activities of miR-NAs. 5,9,15,43 The A→G polymorphism (rs3746444) is located in the stem region outside the mature miR-499-5p sequence. We observed that G-allelic miR-499 precursor displayed a low efficiency to produce mature miR-499-5p compared with the A-allelic one. miR-499 has been shown to suppress the apoptosis through targeting the pro-apoptotic protein CnAα and CnAβ. The A to G variation due to the polymorphism in the miR-499 precursor led to a reduced inhibiting effect of pri-miR-499 on the expression of CnAα/CnAβ and consequently exhibited a less repressive effect on the apoptosis. The Another study has shown that miR-590 C→T SNP (rs6971711) also replaces the stable C-G base pair with wobble U-G base pair and affected the maturation of miR-590 from pre-miR-590. 44 Furthermore, SNPs in the seed region of miRNAs might reduce or even lose their interacting ability with the 3′ UTR of target mRNAs and hence reduce their role of target suppression. 43 Oppositely, it might also change the targets of miRNAs due to increased binding affinity for new target sites. 45 their miRNA content into the circulation upon activation. 49,50 miR-499-5p is a cardiac-specific miRNA and serum miR-499-5p levels are increased at an early time of cardiac injury. miR-499-5p levels were decreased in the plasma of heart failure patients, which may be due to down-regulation of miR-499-5p in the heart under pathological conditions. 22 These results suggested that elevation of miR-499-5p in plasma is may be a result of cardiomyocyte necrosis and subsequent release of cellular constituents into the circulating system in patients with acute MI. It seems that compared with cardiacenriched miRNAs, non-cardiac miRNAs have a comparable potential as MI biomarker. 51,52 However, the origin and release of these miR-NAs in circulation is an interesting query which needs to be investigated in future studies.
It has been reported that the presence of miRNAs in the extracellular space and circulation indicates that these molecules may exhibit signalling functions by acting in an autocrine, paracrine and possibly endocrine manner. 53,54 Although the several studies have assessed the functions of miRNAs in cell-to-cell or cell-to-surrounding-environment communications, however, the consequences and biological effects of systemically released miRNAs on distant sites remain challenging questions. Furthermore, whether the circulating miR-499-5p involved in biological effect at distant sites or in organs or tissues other than the heart need to be further explored.
In summary, this study revealed that miR-499-5p polymorphism may affect its function by regulating apoptosis. Identifying more miRNA polymorphisms and exploring their mechanisms will greatly improve our knowledge of the aetiology of diseases and provide a useful tool for creating an efficient individualized treatment for the complicated diseases.