MiR-216a-5p靶向TRIP4对乳腺癌细胞放射敏感性的影响及机制研究  

Effect of miR-216a-5p targeting TRIP4 on radiosensitivity of breast cancer cells and its mechanism

作　　者：杨霞[1] 汤山松 谢仲梅 张群[1] Yang Xia;Tang Shansong;Xie Zhongmei;Zhang Qun(Department of Oncology,Ziyang People's Hospital,Ziyang 641300,China)

机构地区：[1]四川省资阳市人民医院肿瘤科,资阳641300

出　　处：《广西医科大学学报》2021年第1期61-68,共8页Journal of Guangxi Medical University

基　　金：四川省卫生和计划生育委员会科研项目资助(No.16PJ113)。

摘　　要：目的:研究miR-216a-5p对乳腺癌细胞放疗敏感性的影响及其作用机制。方法:培养正常乳腺上皮细胞MCF10A和乳腺癌细胞MCF7、MDA-MB-231、SK-BR-3,用实时荧光定量PCR(qPCR)检测miR-216a-5p和甲状腺激素受体相互作用蛋白4(TRIP4)的表达;将乳腺癌细胞MCF7分为Control组(未做任何处理)、si-con组(转染TRIP4干扰载体阴性对照质粒)、si-TRIP4组(转染TRIP4干扰载体质粒)、miR-con组(转染miR-216a-5p模拟物阴性对照)、miR-216a-5p组(转染miR-216a-5p模拟物)、si-TRIP4+anti-miR-con组(共转染TRIP4干扰载体质粒和miR-216a-5p抑制剂阴性对照)、si-TRIP4+anti-miR-216a-5p组(共转染TRIP4干扰载体质粒和miR-216a-5p抑制剂);用Western blotting检测蛋白的表达;MTT法检测细胞增殖抑制率;流式细胞术检测细胞凋亡率;分别用0 Gy、2 Gy、4 Gy、6 Gy和8 Gy剂量的射线照射Control组、si-con组、si-TRIP4组、miR-con组、miR-216a-5p组、si-TRIP4+anti-miR-con组、si-TRIP4+anti-miR-216a-5p组细胞,用细胞克隆形成实验检测细胞放射敏感性;将TRIP4野生型(TRIP4-WT)和突变型(TRIP4-MT)荧光素酶表达载体分别与miR-con和miR-216a-5p共转染至MCF7细胞中,用双荧光素酶报告基因实验检测细胞荧光活性。结果:与正常乳腺上皮细胞MCF10A相比,乳腺癌细胞MCF7、MDA-MB-231、SKBR-3中TRIP4 mRNA和蛋白表达水平升高,miR-216a-5p表达水平降低(P<0.05)。沉默TRIP4和过表达miR-216a-5p后,细胞周期蛋白D1(Cyclin D1)表达水平降低,半胱氨酸蛋白酶3(Caspase-3)表达水平升高,细胞增殖抑制率显著升高,细胞凋亡率显著升高(P<0.05),射线照射后细胞存活分数降低(P<0.05)。miR-216a-5p与TRIP4-WT共转染的细胞荧光素酶活性显著降低(P<0.05),而miR-216a-5p与TRIP4-MT共转染的细胞荧光素酶活性无明显变化(P>0.05)。沉默TRIP4逆转了抑制miR-216a-5p表达对乳腺癌细胞增殖、凋亡和放射敏感性的作用。结论:过表达miR-216a-5p可能通过下调TRIP4抑制乳腺癌细胞Objective:To study the effect of miR-216a-5p on the sensitivity of breast cancer cells to radiotherapy and its mechanism.Methods:Culture normal breast epithelial cells MCF10A and breast cancer cells MCF7,MDA-MB-231,and SK-BR-3.qRT-PCR was used to detect the expression of miR-216a-5p and TRIP4.Breast cancer cells MCF7 were divided into control group(no treatment),si-con group(transfected with TRIP4 interference vector negative control plasmid),si-TRIP4 group(transfected with TRIP4 interference vector plasmid),miRcon group(transfected with miR-216a-5p mimic negative control),miR-216a-5p group(transfected miR-216a-5p mimic),si-TRIP4+anti-miR-con group(cotransfected with TRIP4 interference vector plasmid and miR-216a-5p inhibitor negative control),si-TRIP4+anti-miR-216a-5p group(cotransfected with TRIP4 interference vector plasmid and miR-216a-5p inhibitor).Western blotting was used to detect protein expression.MTT method was used to detect cell proliferation inhibition rate.Flow cytometry was used to detect cells apoptosis.The control group,si-con group,si-TRIP4 group,miR-con group,miR-216a-5p group,si-TRIP4+anti-miR-con group and si-TRIP4+anti-miR-216a-5p group cells were irradiated with radiation at doses of 0,2,4,6 and 8 Gy,respectively.Cell clone formation experiment was used to detect cell radiosensitivity.The TRIP4 wild-type(TRIP4-WT)and mutant(TRIP4-MT)luciferase expression vectors were co-transfected with miR-con and miR-216a-5p into MCF7 cells,and the fluorescence activity of the cells was detected by dual luciferase reporter gene experiment.Results:Compared with normal breast epithelial cells MCF10A,the expression levels of TRIP4 mRNA and protein in breast cancer cells MCF7,MDA-MB-231,SK-BR-3 were increased,and the expression of miR-216a-5p was decreased(P<0.05).After silencing TRIP4 and overexpressing miR-216a-5p,the expression of Cyclin D1 was decreased,the expression of Caspase-3 was increased,the cell proliferation inhibition rate was significantly increased,and the apoptosis rate was significantly inc

关 键 词：miR-216a-5p TRIP4 乳腺癌 放射敏感性 增殖 凋亡 

分 类 号：R739.97[医药卫生—肿瘤]