lncRNA MEG3调控AMPK-mTOR自噬通路对H_(2)O_(2)诱导的人卵巢颗粒细胞氧化应激损伤的影响

The influence of IncRNA MEG3 on H_(2)O_(2)-induced oxidative stress injury in human ovarian granulosa cells by regulating AMPK-mTOR autophagy pathway

目的 探究lncRNA MEG3对H_(2)O_(2)诱导的人卵巢颗粒细胞氧化应激损伤的影响及潜在机制。方法 体外培养人卵巢颗粒细胞KGN,不同浓度H_(2)O_(2)处理诱导细胞氧化应激损伤。将KGN细胞分为对照组、H_(2)O_(2)组、si-NC组、si-lncRNA MEG3组、AMPK激活剂组和si-lncRNA MEG3+AMPK激活剂组,除对照组外,其余组均使用200μmol/L H_(2)O_(2)处理。qRT-PCR测量lncRNAMEG3表达;CCK-8评价细胞活力;流式细胞术分析细胞凋亡;荧光探针DCFH-DA检测细胞内ROS含量;商品化试剂盒检测细胞MDA、SOD、GSH水平;单丹磺酰尸胺(MDC)染色检测细胞自噬;Western blot测定AMPK-mTOR通路和自噬相关蛋白表达。结果 50、100、200、400μmol/L H_(2)O_(2)均可诱导KGN细胞氧化应激损伤和lncRNAMEG3表达水平上调。与对照组比较,H_(2)O_(2)组KGN细胞活力、SOD、GSH水平以及mTOR蛋白磷酸化水平显著降低,lncRNA MEG3表达水平、凋亡率、ROS含量、MDA水平、MDC阳性细胞率、Beclin 1、LC3Ⅱ/Ⅰ蛋白水平以及AMPK蛋白磷酸化水平显著升高(P<0.05)。与si-NC组比较,si-lncRNAMEG3组KGN细胞活力、SOD、GSH水平以及mTOR蛋白磷酸化水平显著升高,lncRNA MEG3表达水平、凋亡率、ROS含量、MDA水平、MDC阳性细胞率、Beclin1、LC3Ⅱ/Ⅰ蛋白水平以及AMPK蛋白磷酸化水平显著降低(P<0.05)。si-lncRNA MEG3+AMPK激活剂组KGN细胞凋亡率、ROS含量、MDA水平、MDC阳性细胞率、Beclin 1、LC3Ⅱ/Ⅰ蛋白水平以及AMPK蛋白磷酸化水平显著高于si-lncRNAMEG3组,而显著低于AMPK激活剂组(P<0.05);细胞活力、SOD、GSH水平以及mTOR蛋白磷酸化水平显著低于si-lncRNAMEG3组,而显著高于AMPK激活剂组(P<0.05)。结论 lncRNA MEG3可能通过调控AMPK-mTOR通路抑制自噬,减轻H_(2)O_(2)诱导的人卵巢颗粒细胞氧化应激损伤。

Objective To investigate the influence of lncRNA MEG3 on H_(2)O_(2)-induced oxidative stress injury in hu-man ovarian granulosa cells and its possible mechanism.Methods Human ovarian granulosa cells KGN were cultured in vitro and treated with different concentrations of H2O2 to induce oxidative stress injury.KGN cells were divided into the control group,H_(2)O_(2) group,si-NC group,si-lncRNA MEG3 group,AMPK activator group and si-lncRNA MEG3+AMPK activator group.Ex-cept for the control group,the other groups were treated with 200μmol/L H_(2)O_(2) treatment.The expression level of lncRNA MEG3 was detected by qRT-PCR.CCK-8 was applied to detect the cell viability.Flow cytometry was applied to detect apoptosis.The content of intracellular ROS was detected by fluorescent probe DCFH-DA.Commercial kits were used to detect the level of cel-lular MDA,SOD and GSH.Monodansylcadaverin(MDC)staining was applied to detect autophagy.The expression of AMPK-mTOR pathway and autophagy-related protein were detected by Western blot.Results 50,100,200,and 400μmol/L H_(2)O_(2) could induce oxidative stress injury of KGN cells and up-regulate the expression of lncRNA MEG3.Compared with the control group,the KGN cell viability,the level of SOD and GSH,and the phosphorylation level of mTOR protein in the H2O2 group were significantly decreased,the expression level of lncRNA MEG3,apoptosis rate,the content of ROS,the level of MDA,MDC positive cell rate,the protein level of Beclin 1 and LC3II/I,and the phosphorylation level of AMPK protein were signifi-cantly increased(P<0.05).Compared with the si-NC group,the KGN cell viability,the level of SOD and GSH,and the phos-phorylation level of mTOR protein in the si-lncRNA MEG3 group were significantly increased,the expression level of IncRNA MEG3,apoptosis rate,the content of ROS,level of MDA,MDC positive cell rate,the protein level of Beclin 1 and LC3II/I,and the phosphorylation level of AMPK protein were significantly decreased(P<0.05).Apoptosis rate,the content of ROS,the level of MDA,MDC positive cell rate,the protein level of Beclin 1 and LC3II/I,and the phosphorylation level of AMPK protein of KGN cells in the si-lncRNA MEG3+AMPK activator group were significantly higher than those in the si-lncRNA MEG3 group,and significantly lower than those in the AMPK activator group(P<0.05).The cell viability,the level of SOD and GSH,and the phosphorylation level of mTOR protein were significantly lower than those in the si-lncRNA MEG3 group,and significantly higher than those in the AMPK activator group(P<0.05).Conclusion IncRNA MEG3 may inhibit autophagy by regulating the AMPK-mTOR pathway,and attenuate H2O2-induced oxidative stress injury in human ovarian granulosa cells.