PMS2通过ERK_ERCC1通路对结肠癌SW480细胞生物学行为的影响.pdf

第 49 卷 第 4 期2023年 7 月吉林大学学报（医学版）Journal of Jilin University（Medicine Edition）Vol.49 No.4Jul.2023DOI：10.13481/j.1671587X.20230414PMS2通过 ERK/ERCC1通路对结肠癌 SW480细胞生物学行为的影响黄雪茹1,丁绪浩1,陈素贤2,谭琦2,吴月明3,牛晓敏1,王亚帝4,佟青1（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）摘要 目的目的：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（P0.05或 P0.01），侵袭细胞数明显增加（P0.01），顺铂作用后细胞凋亡率明显降低（P0.01）；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（P0.01），侵 袭 细 胞 数 明 显 减 少（P0.01），顺 铂 作 用 后 细 胞 凋 亡 率 明 显 升 高（P0.01）。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（P0.05 或 P0.05）。与 siRNA-NC 组比较，PMS2 敲减组细胞中 PMS2、ERK1/2 和 p-ERK1/2 蛋白表达水平均明显降低（P0.05 或 P0.01）；与 PMS2 control组比较，PMS2过表达组细胞中 PMS2、ERK1/2和 p-ERK1/2蛋白表达水平均明显升高（P0.01）。结论结论：PMS2表达可影响结肠癌 SW480细胞增殖、迁移、侵袭和抗凋亡能力。PMS2与 ERCC1存在互相作用关系，并可通过调节 ERCC1参与 ERK信号转导通路。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；信号通路中图分类号 R735.35文献标志码 文章编号 1671587X(2023)04093110收稿日期 20220720基金项目 辽宁省教育厅科学研究经费项目（JYTJCZR2020071）作者简介 黄雪茹（1992），女，辽宁省丹东市人，在读硕士研究生，主要从事临床检验方面的研究。通信作者 佟青，教授，主任检验师，硕士研究生导师（E-mail：）；王亚帝，副教授（E-mail：）931第 49 卷 第 4 期 2023 年 7 月吉林大学学报（医学版）Effect of PMS2 on biological behaviors of colon cancer SW480 cells through ERK/ERCC1 pathwayHUANG Xueru1,DING Xuhao1,CHEN Suxian2,TAN Qi2,WU Yueming3,NIU Xiaomin1,WANG Yadi4,TONG Qing1（1.Department of Clinical Laboratory Diagnostics，Third Affiliated Hospital，Jinzhou Medical University，Jinzhou 120001，China；2.Department of Pathology，Third Affiliated Hospital，Jinzhou Medical University，Jinzhou 120001，China；3.Department of Oncology，Third Affiliated Hospital，Jinzhou Medical University，Jinzhou 120001，China；4.Precision Medicine Center，Third Affiliated Hospital，Jinzhou Medical University，Jinzhou 120001，China）ABSTRACT 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 over-expression 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（P0.05 or P0.01），the number of invasion cells was increased（P0.01），the apoptotic rate was decreased after cisplatin treatment（P0.05）；compared with PMS2 control group，the cell proliferative activity and cell migration rate in PMS2 over-expression group were decreased（P0.05），the number of invasion cells was decreased（P0.01），and the apoptotic rate was increased after cisplatin treatment（P0.05）.The protein-protein interaction（PPI）enrichment P value was 2.09e-07，including a total of 13 interaction nodes such as ERC