miR0-34a通过调控SESN2表达促进耳蜗毛细胞凋亡的机制

目的 探讨miR-34a通过调控SESN2的表达促进耳蜗毛细胞（HEI-OC1）凋亡的机制。方法 双萤光素酶报告基因实验检测miR-34a与SESN2靶向相关性；分别构建对照组（NC组）、SESN2过表达组（SESN2组）、共转染SESN2过表达载体及miR-34a mimic组细胞（SESN2+miR 34a mimic组）。应用CCK-8、Annexin V-FITC/PI流式细胞术和ELISA实验分别检测各组HEI-OC1细胞增殖、凋亡及细胞内氧化应激因子表达的变化，WB检测凋亡相关蛋白Bcl-2、Bax与caspase-3及AMPK信号通路中AMPK磷酸化及SIRT1蛋白表达的变化。结果 共转染miR-34a mimic与pGL3-PIK3CA-SESN2-WT后，细胞内萤光素酶活性受到显著抑制；与NC组相比，SESN2组细胞的增殖能力明显增加，而凋亡率显著降低，而SESN2+miR-34a mimic组细胞的增殖及凋亡均无明显变化，提示过表达miR-34a会削弱SESN2对HEI-OC1细胞的保护作用。同时，与NC组相比，SESN2组细胞中凋亡抑制蛋白Bcl-2与SIRT1、p-AMPK/AMPK蛋白相对表达量增加，抑制凋亡蛋白Bax、caspase-3表达，同时细胞内抗氧化应激因子SOD、CAT及GSH-Px表达量随SESN2增加而升高，MDA表达量减少，反之共转染miR-34a mimic能够逆转上述分子表达变化。结论 miR-34a可能通过靶向抑制SENS2表达，促进AMPK/SIRT1信号通路激活、加速细胞内氧化损伤发挥对HEI-OC1细胞凋亡的促进作用。

Molecular mechanism of miR-34a in facilitating cochlear hair cell apoptosis by modulating SESN2 expression

Objective To investigate the molecular mechanism of miR-34a in facilitating cochlear hair cell (HEI-OC1) apoptosis by modulating SESN2 expression.Methods Dualluciferase reporter gene assay detected the targeted correlation between SESN2 and miR-34a. The negative control group (NC), miR-34a-overexpression group (miR-34a mimic), SESN2-overexpression group (SESN2) and the cotransfected group (SESN2+miR-34a mimic) cell lines were constructed respectively. qRT-PCR was used to detect the correlation between miR-34a and SESN2 in HEI-OC1 cells. CCK-8 was proposed to detect the changes in cell proliferation ability. The cell apoptotic rates were measured by flow cytometry.ELISA was used to detect the expression of SOD, CAT, GSH-Px and MDA while the expression of AMPK-related signaling factors were detected by WB. Results Dual-luciferase reporter assay showed that the luciferase activity was significantly inhibited after cotransfected with miR-34a mimic and pGL3-PIK3CA-ESN2-WT plasmids. The expression of miR-34a and SESN2 in HIE-OC1 cells were mutually inhibited. Overexpression of SESN2 significantly promote HEI-OC1 cell proliferation, suppress apoptotic rates and increased the intracellular level of antioxidative stress when compared with the NC group. Conversely, miR-34a-overexpression will weaken the protective effect of SESN2 on HEI-OC2 cells. Overexpression of SESN2 also promotes the relative expression of the anti-apoptotic proteins Bcl-2 and SIRT1, p-AMPK while the expression of apoptotic proteins Bax, caspase-3 were suppressed. The intracellular antioxidant stress factors SOD, CAT and GSH-Px increased with the increase of SESN2 while the expression of MDA was decreased. Conversely, cotransfection with miR 34a mimic reverse the above mentioned molecular expression changes. Conclusion miR-34a may activate AMPK/SIRT1 signaling pathway and accelerate intracellular oxidative damage by targeted inhibition of SENS2 which cause the apoptosis of HEI-OC1 cells.