The MiRNA-30 Family Protects Myocardial Ischemic Injury By Regulating The Expression Of CSE

Objectives:Myocardial infarction can lead to heart tissue damage and a series of pathological changes. MicroRNAs (miRNAs) are approximately 22-nucleotide non-coding RNAs that regulate gene expression by inhibiting mRNA translation or promoting mRNA degradation. Hydrogen sulfide (H2S) is a new gaseous signal molecule regulating cardiovascular function. H2S is mainly produced by cystathionine-y-lyase (CSE) in the cardiovascular system. Recent studies have found that miRNA and H2S are both involved in the protection of myocardial ischemic injury. However, the relationship between miRNA and H2S generation is not clear, and little is known about the role of miRNA in regulating the expression of CSE. The purpose of this study is to find out the miRNAs which can regulate the generation of endogenous H2S, and clarify the role of the miRNA in protecting myocardial ischemic injury by regulating the production of H2S.Methods:1. In order to find the putative miRNA which can regulate the expression of CSE in heart, miRNA prediction analysis software was used, such as TargetScan, miRanda, miTarget etc. Then experimental myocardial infarction models were induced by the ligation of left anterior descending coronary artery in rat and mouse. According to the prediction results, TaqMan real time-PCR was used to determine the miRNA levels in different murine hearts zone including infarct zone, border zone and non-infarct zone and the aberrant expressions of these miRNAs were confirmed. And then, the concentration of H2S in tissue of infarct zone, border zone and non-infarct zone were measured, meanwhile the changes of CSE mRNA and protein expressions were detected by real time-PCR and western blot, respectively. According to these results, analyzed whether the changes of CSE expression, H2S concentration, and miRNA expression were corresponding or not and validated the relationship between miRNA and CSE.2. CSE 3’-UTR was amplified by PCR from Rat genomic DNA and inserted into the pMIR-REPORTTM luciferase miRNA expression reporter vector, then dual luciferase assay was performed to confirm weather CSE transcripts represented a genuine target of miRNA.3. Gain-of-function and loss-of function approaches were employed in cultured acutely isolated neonatal rat myocardial cells. MiRNA mimics and inhibitors were transfected by lipofectamin RNAiMAX.1) The changes of CSE mRNA and protein expressions were detected 48h after transfection by real time-PCR and western blot to determine the regulation of miRNA on CSE expression; 2) The concentrations of H2S in the supernatant were measured after transfecting for 48 hours to validate the effect of miRNA on the production of H2S:3) Cells were incubated in a 3-gas hypoxic chamber maintained at 5% CO2,94%N2 and 1%O2 (oxygen expelled by nitrogen) for 8 hours to induce hypoxia cell model after transfecting for 48 hours. MTT and TUNEL assay were performed to test the cell viability and apoptosis, meanwhile the concentrations of LDH in the supernatant were detected to find out the effect of miRNA mimics and inhibitors on hypoxia injury. These results combined with the change of H2S concentration in the supernatant will confirm the effect of miRNA on hypoxia-induced cell injury after regulating the generation of H2S.4. To further evaluate the biological role of miRNA on myocardial infarction, LNA modified miRNA inhibitor was generated and injected into C57 mouse by tail vein. The downregulation of miRNA in heart tissue was detected by TaqMan real time-PCR after 3 consecutive days’injection. Furthermore, these hearts treated with miRNA inhibitor for 3 consecutive days were subjected to coronary occlusion to establish MI model.1) Then infarct size was measured by TTC and Evans blue double staining to evaluate the effect of LNA-miRNA inhibitor on myocardial infarction.2) The change of heart function was evaluated by Echocardiography (Ejection fraction EF, left ventricular end-systolic volume LVESV, Left ventricular end diastolic volume LVEDV); 3) The change of CSE protein expression in left ventricle was detectd by western blot, meanwhile, the change of H2S concentration in serum was measured to determine the regulation of LNA-miRNA inhibitor on CSE expression and H2S generated; 4) The concentrations of LDH, CK and cTn-I in serum were detect to confirm the effect of LNA-miRNA inhibitor on myocardial infarction; 5) The expressions of Bcl-2 and Bax in left ventricle were detected by western blot to confirm the effect of LNA-miRNA inhibitor on myocardial apoptosis.Results:1. The concentration of H2S in plasma and different rat heart zone including infarct zone, border zone and non-infarct zone were reduced significantly in Rat MI model 48 hours after MI induction. The expressions of CSE mRNA and protein were decreased in border zone, non-infarct zone, but increased in infarct zone which could be a "compensatory effect" to produce H2S to cope with the damage of ischemia.2. MiRNA target gene prediction software (TargetScan Human 6.2) inferred that CSE was a target gene of miR-30 family members including miR-30a, miR-30b, miR-30c, miR-30d, miR-30e; In infarct border zone and non-infarct zone of rat heart which ischemia for 2 days, the TaqMan real time-PCR results showed that miR-30 family members were abnormal expression and all had a tendency to increase. But in infarct area, the expressions of miR-30 family members were reduced significantly.3. The expression of CSE mRNA and protein in primary neonatal rat myocardial cells were reduced significantly after transfecting miR-30 family members mimics for 48h, but increased significantly in a dose-dependent way after transfecting LNA-miR-30 family inhibitor for 48h.4. Immunofluorescence showed the fluorescence intensity was weaker after transfecting with miR-30 family members mimics, indicating the expression of CSE protein was reduced, but the fluorescence intensity became strong after transfecting with LNA-miR-30 family inhibitor.5. Dual luciferase assay results showed miR-30 family members mimics inhibited the activity of the luciferase significantly, indicating miR-30 family memebers could act directly on the 3’-UTR of CSE, CSE was a target gene of miR-30 family memebers.6. MiR-30 family mimics reduced the generation of H2S in the cell supernatant; however, LNA-miR-30 family inhibitor could significantly increase the H2S content in the cell supernatant.7. After hypoxia for 8h, miR-30 family mimics significantly reduced the vitality of the primary cardiomyocytes, increased LDH leakage rate, increased the number of apoptosis induced by hypoxia; LNA-miR-30 family inhibitor could improve the primary cardiomyocytes vitality, reduced LDH leakage rate and hypoxia-induced apoptosis.8. Injection of LNA-miR-30 family inhibitor (5mg/kg) into C57 mouse by tail vein for three consecutive days inhibited the expression of miR-30 family members effectively in vivo; And LNA-miR-30 family inhibitor increased the expression of CSE mRNA and protein in dose-dependent way in mouse heart, and reached the best result in the dose of 2.5mg/kg.9. Compared with the saline group and scrambled NC group, injection of LNA-miR-30 family inhibitor significantly decreased infarct size; reduced plasma CK, LDH and cTn-I content; improved the heart function of mice after myocardial infarction, significantly decreased left ventricular systolic and diastolic diameter, reduced left ventricular end-systolic and end-diastolic volume, at the same time significantly increased ejection fraction and fractional shortening.10. LNA-miR-30 family inhibitor increased H2S levels in plasma and increased the expression of CSE in left ventricle of MI model.11. LNA-miR-30 family inhibitor increased the expression of Bcl-2 in left ventricle and decreased the expression of Bax, thereby reduced myocardial cells apoptosis in left ventricular.Conclusion:1. MiR-30 family members have a direct regulation effect on CSE 3’-UTR, CSE is a target gene of miR-30 family.2. miR-30 family plays a regulatory role in primary neonatal rat myocardial cells during hypoxia injury by regulating the expression of CSE and the generation of H2S. Overexpressions of miRNA-30 family members aggregate hypoxia-induced myocardial cell injury. However, inhibition of the miRNA-30 family can protect myocardial cells against hypoxia injury.3. Inhibition of miR-30 family expression in heart can protect against mouse cardiac ischemic injury by increasing CSE expression and H2S production...