摘要
目的:观察顺铂处理人卵巢颗粒细胞KGN后,细胞中脂肪和肥胖相关基因(FTO)的表达水平和细胞凋亡的变化,探讨FTO基因对化疗后KGN细胞凋亡的调控。方法:分别使用0、5、10、15、20μmol/L顺铂处理KGN细胞24、48、72 h, CCK-8法分析细胞增殖能力变化,流式细胞术检测处理48 h后细胞的凋亡水平,Western blot(WB)法检测细胞中FTO、Bcl-2和Bax蛋白的表达水平。KGN细胞转染针对FTO基因的小干扰RNA(siRNA)(siFTO组),并设立细胞对照组(Control组,只含有脂质体)和siRNA对照组(siNC组,转染阴性对照序列);KGN细胞转染FTO真核表达质粒(FTO组),并设立细胞对照组(Control组,只含有脂质体)和空载体对照组(Vector组,转染空载体)。转染后,同时使用5μmol/L顺铂处理细胞48 h;分别采用qRT-PCR和WB法检测各组FTO、Bcl-2和Bax的表达水平。结果:随着顺铂浓度的增加,各处理时间组KGN细胞的增殖能力均逐渐下降(P<0.05)。细胞凋亡水平随着顺铂浓度的增加逐渐升高(P<0.05),FTO的表达水平逐渐下降(P<0.05)。使用siRNA下调FTO后,抑凋亡蛋白Bcl-2的表达水平下降(P<0.001),促凋亡蛋白Bax的表达水平升高(P<0.001);使用真核表达质粒上调FTO后,抑凋亡蛋白Bcl-2的表达升高(P<0.01),促凋亡蛋白Bax的表达下降(P<0.001)。结论:顺铂能够下调KGN细胞中FTO的表达水平,并通过抑制抑凋亡蛋白Bcl-2表达和上调促凋亡蛋白Bax表达,促进KGN细胞的凋亡,提示化疗可能通过FTO表达促进卵巢颗粒细胞凋亡,引起卵巢早衰。
Objective:To observe the expression of fat mass and obesity-associated protein(FTO)and the level of apoptosis in human ovarian granulocytes treated with cisplatin for investigating the effect of FTO gene on granulocyte apoptosis after chemotherapy.Methods:KGN cells were treated with cisplatin by concentration at 0,5,10,15 and 20μmol/L for 24,48 and 72 h,respectively.The changes in cell proliferation ability were analyzed using CCK-8 method,the level of apoptosis was detected by flow cytometry after 48 hours of treatment.Western blot was used to measure the expression levels of FTO,Bcl-2 and Bax proteins in the cells.KGN cells were transfected with small interfering RNA(siRNA)targeting the FTO gene(siFTO group),and a cell control group(Control group,only containing liposomes)and a siRNA control group(siNC group,transfected with negative control sequences)were established.KGN cells were then transfected with FTO eukaryotic expression plasmids(FTO group),and a cell control group(Control group,only containing liposomes)and an empty vector control group(Vector group,transfected with empty vectors)were established.After transfection,cells were simultaneously treated with 5μmol/L cisplatin for 48 h.finally,the expression levels of FTO,Bcl-2,and Bax in each group were detected using qRT-PCR and Western blot.Results:The proliferation ability of KGN cells gradually decreased in each treatment time group with added concentration of cisplatin(P<0.05),and the level of apoptosis was gradually increased with cisplatin concentration addition(P<0.05),yet FTO expression was gradually downregulated(P<0.05).The relative expression levels of Bcl-2 decreased and Bax were increased after downregulating FTO using siRNA(both P<0.001),and the relative expression levels of Bcl-2 was increased(P<0.01),yet Bax was decreased after upregulating FTO using eukaryotic expression plasmids(P<0.001).Conclusion:Cisplatin can downregulate the expression level of FTO in KGN cells and promote apoptosis of KGN cells by inhibiting the expression of apop
作者
何晶晶
洪名云
戴志俊
朱凯
刘炯炯
HE Jingjing;HONG Mingyun;DAI Zhijun;ZHU Kai;LIU Jiongjiong(Reproductive Medicine Center,Hefei Maternal and Child Health Hospital,Hefei 230001,Anhui,China)
出处
《皖南医学院学报》
CAS
2024年第5期418-421,425,共5页
Journal of Wannan Medical College
基金
安徽省卫生健康委科研项目立项项目(AHWJ2021b127)
安徽省级临床重点专科建设项目(皖卫函2023-320号)。
关键词
卵巢早衰
肥胖相关蛋白
卵巢颗粒细胞
小干扰核糖核酸
细胞凋亡
premature ovarian failure
fat mass and obesity-associated protein
ovarian granulosa cells
small interfering RNA
cell apoptosis
