miR-34a通过调控SESN2表达促进耳蜗毛细胞凋亡的机制

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

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。

Objective To investigate the molecular mechanism of miR-34a in facilitating cochlear hair cell(HEI-OC1)apoptosis by modulating SESN2 expression.Methods Dual-luciferase 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 co-transfected 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 co-transfected with miR-34a mimic and pGL3-PIK3CA-SESN2-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 anti-oxidative 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,co-transfection 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.