METTL3介导的LncRNA MIR99AHG通过抑制miR-136-5p表达诱导卵巢颗粒细胞凋亡的机制研究  

Study on the mechanism of METTL3-mediated LncRNA MIR99AHG inducing ovarian granulosa cell apoptosis by inhibiting the expression of miR-136-5p

作　　者：常建芳 孙延庆 Chang Jianfang;Sun Yanqing(Department of Reproductive Medicine,960th Hospital of PLA Joint Logistic Support Force,Jinan Shandong 250000,P.R.China)

机构地区：[1]联勤保障部队解放军第960医院生殖医学科,山东济南250000

出　　处：《中国计划生育和妇产科》2024年第6期58-64,共7页Chinese Journal of Family Planning & Gynecotokology

基　　金：山东省自然科学基金资助项目(项目编号:ZR2021HM062)。

摘　　要：目的 研究LncRNA MIR99AHG(MIR99AHG)在化疗药物导致的卵巢颗粒细胞凋亡中的作用及其潜在的调节机制。方法 采集正常女性和卵巢早衰(premature ovarian failure, POF)患者的卵泡液,检测MIR99AHG表达。将卵巢颗粒细胞KGN分为对照组、顺铂组、顺铂+NC shRNA组、顺铂+sh-MIR99AHG组、顺铂+vector组、顺铂+OE-METTL3组、顺铂+OE-METTL3+OE-MIR99AHG组、顺铂+OE-MIR99AHG组、顺铂+NC mimic组、顺铂+miR-136-5p mimic组及顺铂+sh-MIR99AHG+NC inhibitor组和顺铂+sh-MIR99AHG+miR-136-5p inhibitor组。KGN细胞经相应载体或质粒处理后,采用RIP分析MIR99AHG的m6A修饰水平;在线数据库预测MIR99AHG与miR-136-5p的结合位点,并通过荧光素酶报告基因分析进行验证;采用CCK-8和流式细胞术分析KGN细胞的活力和凋亡水平;ELISA检测Caspase-3酶活性;Western blot检测凋亡相关蛋白BCL-2和Bax表达水平。结果 与正常女性相比,POF患者卵泡液中MIR99AHG表达水平显著上调(P<0.001)。与对照组相比,顺铂处理组KGN细胞中MIR99AHG表达显著上调,METTL3表达及蛋白水平显著下调(P<0.05)。与对照组相比,顺铂处理组KGN细胞活力显著降低,细胞凋亡率显著升高(P<0.05);而与NC shRNA组相比,sh-MIR99AHG组KGN细胞活力显著升高,细胞凋亡率显著降低(P<0.05)。RIP分析发现,Anti-m6A抗体下拉复合物中MIR99AHG显著富集。与vector组相比,OE-METTL3组MIR99AHG的m6A水平显著升高,MIR99AHG mRNA表达水平显著降低(P<0.05);OE-METTL3组KGN细胞活力显著升高,细胞凋亡率显著降低(P<0.05);共转染OE-MIR99AHG可逆转OE-METTL3对KGN细胞活力、凋亡及相关蛋白表达的影响。miR-136-5p与MIR99AHGA存在结合位点,MIR99AHGA负调控miR-136-5p表达。与NC-mimic组相比,miR-136-5p mimic组细胞活力及BCL-2蛋白表达明显升高,细胞凋亡率、Caspase-3酶活性及Bax蛋白表达明显降低(P<0.05)。共转染miR-136-5p inhibitor逆转了sh-MIR99AHG对细胞KGN细胞活力、凋亡及相关蛋白表达的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-MIR99AHG 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 v

关 键 词：卵巢颗粒细胞 长链非编码mir-99a-let-7c簇宿主基因 N6-甲基腺苷修饰 N6腺苷甲基转移酶样3 微小核糖核酸-136-5p 

分 类 号：R711.75[医药卫生—妇产科学] R34[医药卫生—临床医学]