指环蛋白31对表皮生长因子受体表达的调控作用及其机制  

Regulatory effect of ring finger protein 31 on expression of epidermal growth factor receptor and its mechanism

作　　者：薛富强 苏荣健 贺武斌 宋慧娟 栾治东 XUE Fuqiang;SU Rongjian;HE Wubin;SONG Huijuan;LUAN Zhidong(.Department of Developmental Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China;Department of Cell Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China)

机构地区：[1]锦州医科大学基础医学院发育生物学教研室,辽宁锦州121000 [2]锦州医科大学基础医学院细胞生物学教研室,辽宁锦州121000

出　　处：《吉林大学学报（医学版）》2023年第1期15-21,共7页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(81772648)。

摘　　要：目的:探讨指环蛋白31(RNF31)对表皮生长因子受体(EGFR)表达的影响,并分析RNF31对EGFR的调控机制。方法:采用免疫共沉淀(Co-IP)法验证RNF31蛋白与EGFR蛋白的相互作用。HEK293T细胞分为空载体组(转染pcDNA3.1空载体)和RNF31组(转染RNF31-Flag质粒),采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测细胞中EGFR mRNA和蛋白表达水平。RNF31的泛素连接酶功能位点(RNF31C699/702S)和去泛素化酶结合位点(RNF31N84A Y93A)突变后,采用Western blotting法检测各组细胞中EGFR蛋白表达水平。HEK293细胞分为空载体组、RNF31组(转染RNF31-Flag质粒)、RNF31+EGFR抑制剂吉非替尼(Gefitinib)组和RNF31+EGFR抑制剂厄洛替尼(Erlotinib)组,采用Western blotting法检测各组细胞中EGFR及其下游信号通路蛋白表达水平。结果:Co-IP法检测,RNF31蛋白与EGFR蛋白存在相互作用。与空载体组比较,RNF31组细胞中EGFR mRNA表达水平明显降低(P<0.05)。与空载体组比较,转染RNF31C699/702S组和野生型RNF31组细胞中EGFR蛋白表达水平明显升高(P<0.05)。与RNF31组比较,转染RNF31N84A Y93A组细胞中EGFR蛋白表达水平明显降低(P<0.05)。与空载体组比较,RNF31组细胞中EGFR及其下游信号通路中核因子κB(NF-κB)、磷酸化信号传导与转录激活因子3(p-STAT3)、丝裂原活化蛋白激酶(MAPK)和磷酸化MAPK(p-MAPK)蛋白表达水平明显升高(P<0.05),RNF31+Gefitinib组和RNF31+Erlotinib组细胞中蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、STAT3和MAPK蛋白表达水平明显降低(P<0.05)。结论:RNF31通过去泛素化酶结合结构域发挥去泛素化的作用,降低EGFR蛋白分子表面的泛素化水平,使EGFR蛋白降解速率减慢,同时高表达的EGFR可以激活其下游与细胞增殖和分裂相关蛋白的表达。Objective:To investigate the effect of ring finger protein 31(RNF31)on the expression of epidermal growth factor receptor(EGFR),and to explore the regulation mechanism of RNF31 on EGFR.Methods:The interaction between RNF31 and EGFR proteins was validated by co-immunoprecipitation(Co-IP)method.The HEK293T cells were divided into empty vector group(transfcted with pcDNA3.1 emptey vector)and RNF31 group(transfected with RNF31-Flag plasmid).The expression levels of EGFR mRNA and protein in the cells were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods.After mutation of the ubiquitin ligase function of RNF31(RNF31C699/702S)and deubiquitinase binding site(RNF31N84A Y93A),Western blotting method was used to detect the expression levels of EGFR protein in the cells in various groups.The HEK293T cells were divided into empty vector group,RNF31 group(transfected RNF31-Flag plasmid),RNF31+EGFR inhibitor Ggfitinib(Gefitinib)group and RNF31+EGFR inhibitor Erlotinib(Erlotinib)group;the expression levels of EGFR and its downstream sigaling pathway proteins in the cells in various groups were detected by Western blotting method.Results:The Co-IP method detection results confirmed that the RNF31 was interacted with EGFR.Compared with empty vector group,the expression level of EGFR mRNA in the cells in RNF31 group was significantly decreased(P<0.05).Compared with empty vector group,the expression levels of EGFR protein in the cells in RNF31C699/702S group and wild-type RNF31 group were significantly increased(P<0.05).Compared with RNF31 group,the expression level of EGFR protein in the cells in transfection RNF31N84A Y93A group was decreased(P<0.05).Compared with empty vector group,the expression levels of EGFR and nuclear transcription factor-κB(NF-κB),phosphorylated signal transducer and activiator of transcription 3(p-STAT3),mitogen-activated protein kinase(MAPK)and phosporylated MAPK(p-MAPK)proteins in its downstream signling pathways in the cells in RNF31 group were significantly inc

关 键 词：指环蛋白31 表皮生长因子受体 泛素化 蛋白互作 去泛素化酶 

分 类 号：Q78[生物学—分子生物学]