Study on the mechanism of METTL3-mediated LncRNA MIR99AHG inducing ovarian granulosa cell apoptosis by inhibiting the expression of miR-136-5p
Objective To investigate the role of LncRNA MIR99AHG(MIR99AHG)in chemotherapy-induced apoptosis of ovarian granulosa cells,and its potential regulatory mechanisms.Methods The follicular fluid of normal women and premature ovarian failure(POF)patients were collected to detect the expression of MIR99AHG.KGN cells and a cell line of ovarian granulosa cells were divided into the control group,cisplatin group,cisplatin+NC shRNA group,cisplatin+sh-MIR99AHG group,cisplatin+vector group,cisplatin+OE-METTL3 group,cisplatin+OE-METTL3+OE-MIR99AHG group,cisplatin+OE-M1R99AHG group,cisplatin+NC mimic group,cisplatin+miR-136-5p mimic group,cisplatin+sh-MIR99AHG+NC inhibitor group and cisplatin+sh-MIR99AHG+miR-136-5p inhibitor group.After KGN cells were treated with corresponding vectors or plasmids,the m6A modification levels of MIR99AHG was analyzed by RIP.The binding of MIR99AHG to its target miR-136-5p was predicted by online database and validated by the luciferase reporter gene assay.Furthermore,the viability and apoptosis of KGN were analyzed by CCK-8 and flow cytometry;Caspase-3 activity was detected with ELISA;and the expression levels of apoptosis related proteins BCL-2 and Bax were detected with Western blot.Results Compared with normal women,the expression of MIR99AHG in the follicular fluid of POF patients was significantly up-regulated(P＜0.001).Compared with the control group,the expression of MIR99AHG in KGN cells in cisplatin treated group was significantly up-regulated,and the expression and protein level of METTL3 were significantly down-regulated(P＜0.05).Moreover,compared with the control group,the viability of KGN cells in the cisplatin treatment group was significantly decreased,and cell apoptosis was significantly increased(P＜0.05);Compared with NC shRNA group,the viability of KGN cells in sh-MIR99AHG group was significantly increased and cell apoptosis was significantly decreased(P＜0.05).RIP analysis showed that MIR99AHG was significantly enriched in the Anti-m6A antibody pull-down complex.Compared with vector group,the m6A level of MIR99AHG in OE-METTL3 group was significantly increased,and the MIR99AHG mRNA expression level was significantly decreased(P＜0.05);The viability of KGN cells in OE-METTL3 group was significantly increased,and the apoptosis rate was significantly decreased(P＜0.05).Co-transfection of OE-MIR99AHG reversed the effects of OE-METTL3 on KGN cell viability,apoptosis and expression of related proteins.There was a binding site between miR-136-5p and MIR99AHGA,and MIR99AHGA negatively regulated the expression of miR-136-5p.Compared with NC-mimic group,the cell viability and BCL-2 protein expression of miR-136-5p mimic group were significantly increased,while the cell apoptosis rate,Caspase-3 activity and Bax protein expression were significantly decreased(P＜0.05).Co-transfection with miR-136-5p inhibitor reversed the effects of sh-MIR99AHG on KGN cell viability,apoptosis and related protein expression(P＜0.05).Conclusion Cisplatin induces the upregulation of MIR99AHG in ovarian granulosa cells.METTL3 inhibited the expression of MIR99AHG through upregulating of m6A levels,to promote the upregulation of miR-136-5p,thereby inhibiting the apoptosis of ovarian granulosa cells and improving chemotherapy drug cisplatin mediated premature ovarian failure.
ovarian granulosa cellslong-chain non coding mir-99a-let-7c cluster host genes(MIR99AHG)N6-methyladenosine(m6A)modificationN6 adenosine methyltransferase like 3(METTL3)micro-ribonucleic acid(miR)-136-5p
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