The Study On The Role And Mechanism Of MiR-208a Against Myocardial Ischemia-reperfusion Injury

BackgroundCurrently, open-heart surgery is widely used to treat various types of heart diseases, and cardiopulmonary bypass (CPB) is an essential component of open-heart surgery. However, a few studies have revealed that the surgery itself may result in intra-operative injury to multiple organs, especially the heart, with ischemia-reperfusion (I/R) injury being the most common.. MicroRNAs (miRNAs) are single-stranded, short-length (21 to 23 nucleotides), noncoding RNAs that act as negative posttranscriptional modulators of target mRNAs. miRNAs play a major regulatory role in a myriad of mechanisms in developmental biology, physiology, and pathology of almost every organ, including those within the cardiovascular system. Increasing evidence indicates that miRNAs may play roles in the development of I/R injury.AimTo measure the alteration of several miRNA expression levels in open-heart surgery with cardiopulmonary bypass (CPB), and to further investigate role of miR-208a in ischemia reperfusion (I/R) injury. The present study provide a theoretical basis for the early diagnosis, prevention and treatment of I/R injury, also provide a potential molecular target for future therapyMethod1, Creatine kinase (CK-MB), cardiac troponin (cTnI) and plasma miRNAs (miR-1/21/208a/499) were measured at different times during open-heart surgery for combined mitral and aortic valve replacement (n=15 patients). Selected miRNAs were measured at pre-surgery,45 min after aortic-clamping,60 min after reperfusion and 24 h post-surgery using quantitative RT-PCR.2, To construct the I/R model mice, and harvest the hearts from two group mice. Fifteen mices in the control group (Sham) was only open the heart, and another fifteen mices in the study group (I/R) was lighted of left anterior descending. The total RNA was extracted and the expression of miR-208a was measured using RT-qPCR. Myocardial infarction range was sacrificed to get Evans blue stain and triphenyl tetrazolium chloride (TTC). The apoptosis rate of cardiac cells was detected by TUNEL assay. Meanwhile, caspase-3 was determined by western blot.3, The antagomir (anta-miR-NC and anta-miR-208a) was injected into the mices by the tail vein for two days, respectively, and the mices without injecting antagomir were as the control. Then ischemia and reperfusion procedure was performed in both groups. After 24 hours, they were sacrificed to get TTC-Phthalocyanine, blue staining or western blot. The myocardial tissue morphology was detected by HE staining. The apoptosis rate of cardiac cells was detected by TUNEL. Meanwhile, caspase-3 was determined by western blot. The atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) were also detected using RT-qPCR, and LDH levels of mice from different group were measured in assessing myocardial function.4, The mRNA, target by miR-208a, were predicted using bioinformatics method, and then were validated by dual luciferase report experiment, And after transfection using flow cytometry apoptosis of cells in each group.Results1, Significant differences were found in miR-1/208a/499 levels before CPB and after reperfusion; no such difference was found in miR-21 levels. Specifically, miR-1/208a levels remained unchanged until 45 min after aortic-clamping, when they increased 60 min after reperfusion (P<0.05) then fell 24 h post-surgery (P<0.05). The changes in miR-1/208a expression levels were similar to those of cTnl and CK-MB, and miR-208a reflected faster than miR-1. In contrast, miR-499 expression levels fell after reperfusion (P< 0.05) and remained down 24h post-surgery. Nevertheless, this expression was negatively correlated with cTnI and CK-MB levels in the samples from all time points.2, The cardiac ischemia reperfusion injury in mices model was successfully constructed..Comparing with the Sham group, the expression levels of miR-208a in the I/R group were significantly higher (P<0.05); Cell apoptosis rates increased significantly (P<0.05) and the expression of apoptosis markers Caspase 3 enhanced (P<0.05). It suggested that miR-208a may have a definite correlation with myocardial cell apoptosis.3, The results showed that the antagomir significantly reduced the expression levels of miR-208a compared to the I/R or I/R+anta-control group (P<0.05). The ratio of the infarct area/risk area was significantly lower in the I/R+anta-miR-208a group (n=8) compared with the other groups (n=8) (P<0.05). Western blot revealed that the expression level of cleaved caspase3 was significantly inhibited, and the apoptosis rate decreased (P<0.05). The levels of ANF, BNF and LDH significantly reduced (P<0.05), which indicated that myocardial dysfunction recovery.4, The predicted results showed that the GATA4 and QKI5 were the candidate target genes of miR-208a. In the initial screening assay, the mRNA expression of GATA4 or QKI5 was significantly lower in the I/R group than that in the Sham group (P<0.05). There was the negative correlation between the levels of miR-208a and GATA4 or QKI5. Therefore, we supposed that GATA4 and QKI5 might be a target gene of miR-208a.Further,luciferase activity in the experimental set was significantly decreased in the control set (P<0.01),confirming that miR-208a mimics repressed the luciferase expression of the pMIR-GATA4 but not pMIR-QKI5. These suggest that GATA4 not QKI5 is a target gene of miR-208a.Conclusion1. miR-1/208a/499 may be sensitive biomarkers and potential therapeutic targets for cardiac protection against I/R during open-heart surgery with CPB.2. In the I/R injury model mice, the expression levels of miR-208a increased, and the apoptosis.3. Inhibition of miR-208a could decrease the myocardial apoptosis, and reduce the myocardial dysfunction induced by ischemia reperfusion, with protect the heart.4. The protecting effect of miR-208a may be involved in targeting GATA4 and regulating QKI5 related pathway.