PMS2通过ERK/ERCC1通路对结肠癌SW480细胞生物学行为的影响  

Effect of PMS2 on biological behaviors of colon cancer SW480 cells through ERK/ERCC1 pathway

作　　者：黄雪茹 丁绪浩 陈素贤 谭琦 吴月明 牛晓敏 王亚帝 佟青 HUANG Xueru;DING Xuhao;CHEN Suxian;TAN Qi;WU Yueming;NIU Xiaomin;WANG Yadi;TONG Qing(Department of Clinical Laboratory Diagnostics,Third Affiliated Hospital,Jinzhou Medical University,Jinzhou 120001,China;Department of Pathology,Third Affiliated Hospital,Jinzhou Medical University,Jinzhou 120001,China;Department of Oncology,Third Affiliated Hospital,Jinzhou Medical University,Jinzhou 120001,China;Precision Medicine Center,Third Affiliated Hospital,Jinzhou Medical University,Jinzhou 120001,China)

机构地区：[1]锦州医科大学附属第三医院临床检验诊断学教研室,辽宁锦州121000 [2]锦州医科大学附属第三医院病理科,辽宁锦州121000 [3]锦州医科大学附属第三医院肿瘤二科,辽宁锦州121000 [4]锦州医科大学附属第三医院精准医学中心,辽宁锦州121000

出　　处：《吉林大学学报（医学版）》2023年第4期931-940,共10页Journal of Jilin University：Medicine Edition

基　　金：辽宁省教育厅科学研究经费项目(JYTJCZR2020071)。

摘　　要：目的:探讨减数分裂后分离蛋白2(PMS2)表达对结肠癌SW480细胞生物学行为的影响,阐明PMS2与切除修复交叉互补组1(ERCC1)和细胞外调节蛋白激酶(ERK)信号转导通路的关系。方法:将PMS2 siRNA质粒和PMS2过表达质粒分别转染入结肠癌SW480细胞(分别为PMS2敲减组和PMS2过表达组),同时设PMS2敲减对照组(siRNA-NC组)和PMS2过表达对照组(PMS2 control组)。采用实时荧光定量PCR(RT-qPCR)法检测各组细胞中PMS2 mRNA表达水平,Western blotting法检测各组细胞中PMS2蛋白表达水平,CCK-8法检测各组细胞增殖活性,细胞划痕实验检测各组细胞迁移率,Transwell小室实验检测各组细胞中侵袭细胞数,流式细胞术检测顺铂作用后各组细胞凋亡率。通过String数据库,对PMS2、ERCC1和ERK上下游蛋白的关系进行生物信息学分析。SW480细胞分别采用3条siRNA进行PMS2和ERCC1敲减,采用RT-qPCR法验证PMS2与ERCC1的相互作用,采用Western blotting法检测各组细胞中PMS2、细胞外调节蛋白激酶1/2(ERK1/2)和磷酸化ERK1/2(p-ERK1/2)蛋白表达水平。结果:RT-qPCR法和Western blotting法检测,PMS2基因敲减和过表达细胞模型构建成功。与siRNA-NC组比较,PMS2敲减组细胞增殖活性和细胞迁移率明显升高(P<0.05或P<0.01),侵袭细胞数明显增加(P<0.01),顺铂作用后细胞凋亡率明显降低(P<0.01);与PMS2 control组比较,PMS2过表达组细胞增殖活性和细胞迁移率明显降低(P<0.01),侵袭细胞数明显减少(P<0.01),顺铂作用后细胞凋亡率明显升高(P<0.01)。蛋白-蛋白互作(PPI)富集P值为2.09e-07,包含ERCC1和ERK1/2等相互作用节点数共有13个,提示PMS2、ERCC1和ERK1/2之间可能存在调控作用。与siRNA-NC组比较,各PMS2敲减组细胞中ERCC1 mRNA表达水平明显降低(P<0.05或P<0.01);与siERCC1-NC组比较,各ERCC1敲减组细胞中PMS2 mRNA表达水平差异无统计学意义(P>0.05)。与siRNA-NC组比较,PMS2敲减组细胞中PMS2、ERK1/2和p-ERK1/2蛋白表达水平均�Objective:To discuss the effect of the expression of post-meiotic segregated protein 2(PMS2)on the biological behaviors of colon cancer SW480 cells,and to clarify the relationships between PMS2 and excision repair cross-complementation group 1(ERCC1)and extracellular regulated protein kinase(ERK)signal transduction pathway.Methods:The PMS2 siRNA and PMS2 over-expression plasmids were respectively transfected into the colon cancer SW480 cells(knockdown group and PMS2 over-expression group).The PMS2 knockdown control group(siRNA-NC group)and PMS2 overexpression control group(PMS2 control group)were wet up.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of PMS2 mRNA in cells in various groups;the expression levels of PMS2 protein in the cells in various groups were detected by Western blotting method;the proliferation activities of cells in various groups were detected by CCK-8 assay;cell scratch test was used to detect the migration rates of cells in various groups;the numbers of invasion cells in various groups were detected by Transwell chamber assay;the apoptotic rates of cells in various groups after cisplatin treatment were detected by flow cytometry;bioinformatics analysis was performed on the relationship between upstream and downstream proteins of PMS2,ERCC1,and ERK by using String Database;the PMS2 and ERCC1 knockdown in the SW480 cells were treated with 3 siRNA.The RT-qPCR method was used to verify the reaction between PMS2 and ERCC1;the expression levels of PMS2,extracellular regulated protein kinases 1/2(ERK1/2),and phosphorylated ERK1/2(p-ERK1/2)proteins in the cells in various groups were detected by Western blotting method.Results:The RT-qPCR and Western blotting results showed that the cell model of PMS2 gene knockdown and over-expression was successfully established.Compared with siRNA-NC group,the cell proliferative activity and cell migration rate in PMS2 knockdown group were increased(P<0.05 or P<0.01),the number of invasion cells was increased(P<0

关 键 词：结肠肿瘤 SW480细胞 减数分裂后分离蛋白2 切除修复交叉互补组1 细胞外调节蛋白激酶1/2 信号通路 

分 类 号：R735.35[医药卫生—肿瘤]