RAD51相关蛋白1在宫颈癌组织中的表达及通过TGF-β/Smad通路参与调控人宫颈癌细胞的增殖和凋亡

Expression of RAD51AP1 in cervical cancer and its regulation on proliferation and apoptosis of human cervical cancer cells through TGF-β/Smad pathway

目的:探讨RAD51相关蛋白1(RAD51AP1)通过调节转化生长因子β(TGF-β)/Smad信号通路对人宫颈癌细胞(SiHa)增殖和凋亡的影响。方法:收集2018年7月至2021年3月间南阳市中心医院51例宫颈癌组织与癌旁组织标本,免疫组织化学法检测宫颈癌组织RAD51AP1蛋白表达。筛选RAD51AP1高表达宫颈癌细胞系,体外培养SiHa细胞,分为对照组、si-RAD51AP1组(转染si-RAD51AP1质粒)、si-NC组(转染si-NC质粒)、抑制剂组(TGF-β/Smad通路抑制剂LY2109761)、si-RAD51AP1+激活剂组(转染si-RAD51AP1质粒+TGF-β/Smad通路激活剂人重组TGF-β1)。采用MTT法与平板克隆形成实验检测各组SiHa细胞增殖;流式细胞术检测细胞凋亡;实时荧光定量PCR法检测细胞中RAD51AP1、TGF-β1 mRNA水平;免疫印迹检测细胞中RAD51AP1蛋白、TGF-β/Smad通路相关蛋白及增殖、凋亡相关蛋白表达。结果:RAD51AP1在宫颈癌组织与SiHa细胞系中均显著高表达;抑制RAD51AP1表达或抑制TGF-β/Smad通路后,细胞增殖活性、细胞克隆形成率及cyclin D1、TGF-β1、p-SMAD2、p-SMAD3蛋白表达水平显著减低,细胞凋亡率、Bax、caspase-3、cleaved caspase 3蛋白表达水平显著升高,而抑制RAD51AP1表达基础上使用TGF-β/Smad通路激活剂后,可部分逆转上述变化。结论:RAD51AP1在宫颈癌中表达上调,抑制RAD51AP1表达可通过抑制TGF-β/Smad通路而抑制宫颈癌细胞增殖,促进细胞凋亡。

Objective:To investigate the effect of RAD51-associated protein 1(RAD51AP1) on the proliferation and apoptosis of human cervical cancer cells(Si Ha) by regulating transforming growth factor β(TGF-β)/Smad signaling pathway.Methods:A total of 51 cervical cancer tissues and paracancerous tissues were collected from Nanyang Central Hospital during July 2018 and March 2021,and the expression of RAD51AP1 protein in cervical cancer tissues was detected by immunohistochemistry.Cervical cancer cell lines with high expression of RAD51AP1 were screened.Si Ha cells were cultured in vitro and were divided into a control group,an si-RAD51AP1 group(transfected with si-RAD51AP1 plasmid),an si-NC group(transfected with si-NC plasmid),an inhibitor group(TGF-β/Smad pathway inhibitor LY2109761),and an si-RAD51AP1+activator group(transfected with siRAD51AP1 plasmid+TGF-β/Smad pathway activator human recombinant TGF-β1).MTT method and plate clone formation experiment were used to detect the proliferation of Si Ha cells in each group.Flow cytometry was used to detect cell apoptosis.RT-q PCR method was used to detect the levels of RAD51AP1 and TGF-β1 m RNA in cells.Western blot was used to detect the expression of RAD51AP1 protein,TGF-β/Smad pathway related proteins,and proliferation and apoptosis related proteins in cells.Results:RAD51AP1 was significantly highly expressed in cervical cancer tissues and cell lines;after inhibiting the expression of RAD51AP1 or inhibiting the TGF-β/Smad pathway,the cell proliferation activity,cell clone formation rate and Cyclin D1,TGF-β1,p-SMAD2,p-SMAD3 proteins expression level was significantly decreased.The apoptosis rate,Bax,caspase-3,and cleaved caspase 3 protein expression levels were significantly increased,the use of TGF-β/Smad pathway activator on the basis of inhibiting the expression of RAD51AP1 could partially reverse this changes.Conclusion:RAD51AP1 is up-regulated in cervical cancer.Inhibiting the expression of RAD51AP1 can inhibit the proliferation of cervical cancer cells and promote cell apoptosis by inhibiting the TGF-β/Smad pathway.