关键词: 心肌细胞, 缺氧/复氧, 微小RNA-320a, Janus激酶2/信号转导因子和转录激活因子3信号通路, 细胞凋亡

Abstract:

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. 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.

Key words: Cardiomyocyte, Hypoxia/reoxygenation, MicroRNA-320a, Janus kinase 2/signal transducers and activator of transcription 3 signaling pathway, Apoptosis