The influence of lncRNA MEG3 on H2O2-induced oxidative stress injury in human ovarian granulosa cells by regulating AMPK-mTOR autophagy pathway
Objective To investigate the influence of lncRNA MEG3 on H2O2-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,H2O2 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 200 μmol/L H2O2 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 400 μmol/L H2O2 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 lncRNA 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 lncRNA MEG3 may inhibit autophagy by regulating the AMPK-mTOR pathway,and attenuate H2O2-induced oxidative stress injury in human ovarian granulosa cells.
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