下调 miR-320a 表达对缺氧/复氧诱导的心肌细胞增殖和 凋亡的影响.

Objective: To discuss the effect of down-regulation of miR-320a expression on the myocardial hypoxia/reoxygenation (H/R)injury model, and to clarify its related mechanism. Methods: Real-time fluorescence quantitative PCR (RT-qPCR)method was used to detect the expression levels of miR-320a in serum of the patients with actue myocardial infarction (AMI)and the myocardial H9C2 cells induced by H/R. The miR-320a inhibitor, inhibitor NC, small interference Janus kinase 2 (si-JAK2), and si-NC plasmids were transfected into the H9C2 cells respectively, and blank control group was set up. After successful transfection, the H/R treatment was performed. The H9C2 cells were divided into control group, H/R group, H/R + inhibitor NC group, H/R + miR-320a inhibitor group, H/R + miR-320a inhibitor + si-NC group and H/R + miR-320a inhibitor + si-JAK2 group. The targeting relationship between miR-320a and Janus kinase 2 (JAK2)was detected by double luciferase reporter gene; the proliferation rate of cells in various groups were detected by CCK-8 assay; the activities of superoxide dismutase (SOD)and levels of malonaldehyde (MDA)in cells and the levels of lactate dehydrogenase (LDH)in cell culture supernanant in various groups were detected by biochemical method; the apoptotic rates of cells in various groups were detected by flow cytometry; the expression levels of miR-320a and JAK2 mRNA in cells in various groups were detected by RT-qPCR method; the expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved-cysteinyl aspartate specific proteinase-3 (cleaved-caspase-3), JAK2, signal transducers and activator of transcription 3 (STAT3), and phosphorylated STAT3 (p-STAT3)proteins in cells in various groups were detected by Western blotting method. Results: The expression levels of miR-320a in serum of the patients with AMI and the myocardial H9C2 cells in H/R group were significantly higher than those in control group (P<0. 05). The results of double Luciferase reporter gene detection suggested that miR-320a could targetedly bind with JAK2. Compared with control group, the proliferation rate of the cells and SOD activity in the cells in H/R group were decreased significantly (P<0. 05), the apoptotic rate of the cells, MDA level and LDH activity in the cells were significantly increased (P<0. 05), the expression levels of Bcl-2 and JAK2 proteins and ratio of p-STAT3/STAT3 in the cells were significantly decreased (P<0. 05), and the expression levels of Bax and cleaved caspase-3 proteins in the cells were significantly increased (P<0. 05). Compared with H/R group, the proliferation rate of the cells and SOD activity in the cells in H/R+miR-320a inhibitor group were increased (P<0. 05), while the apoptotic rate of the cells, MDA level in the cells, LDH activity in the cell culture supernanant were decreased (P<0. 05), the expression levels of Bcl-2 and JAK2 proteins and ratio of p-STAT3/STAT3 in the cells were significantly increased (P<0. 05), the expression levels of Bax and cleaved- caspase-3 proteins in the cells were significantly decreased (P<0. 05). Compared with H/R+miR-320a inhibitor+ si-NC group, the proliferation rate of the cells and SOD activity in the cells in H/R+miR-320a inhibitor+ si-JAK2 group were decreased (P<0. 05), and the apoptotic rate of the cells, MDA level in the cells, and LDH activity in the cell culture supernanant were increased (P<0. 05). Conclusion: Down-regulation of miR-320a expression can inhibit the apoptosis of the cardiomyocytes induced by H/R and increase the proliferation activity of cells, and its mechanism is related to the targeted regulation of JAK2/STAT3 signaling pathway. [ABSTRACT FROM AUTHOR]