脆性X智力障碍蛋白靶向DDX5调控Wnt/β-catenin信号通路促进乳腺癌细胞上皮-间质转化发生  

DDX5-Targeting Fragile X Mental Retardation Protein Regulates the Wnt/β-catenin Signaling Pathway to Promote Epithelial Mesenchymal Transition in Breast Cancer

作　　者：曹佳 王晶 石斌 马小兰 吴伟超 王南 CAO Jia;WANG Jing;SHI Bin;MA Xiaolan;WU Weichao;WANG Nan(Medical Science Research Institute,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Department of Pathology,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Department of Emergency,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Department of Otolaryngology,Yinchuan First People's Hospital,Yinchuan 750004,China;No.2 Department of Oncology,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院医学科学研究院,银川750004 [2]宁夏医科大学总医院病理科,银川750004 [3]宁夏医科大学总医院急诊科,银川750004 [4]银川市第一人民医院耳鼻喉科,银川750004 [5]宁夏医科大学总医院肿瘤内二科,银川750004

出　　处：《四川大学学报（医学版）》2024年第5期1138-1149,共12页Journal of Sichuan University(Medical Sciences)

基　　金：宁夏回族自治区重点研发计划项目(No.2022BEG03124);宁夏医科大学2023年校级项目(No.XM2023023)资助。

摘　　要：目的探讨脆性X智力障碍蛋白(fragile X mental retardation protein,FMRP)促进乳腺癌细胞迁移、上皮-间质转化(epithelial mesenchymal transition,EMT)发生及其可能的作用机制。方法采用RT-PCR、Western blot方法,分析FMRP在正常乳腺上皮细胞(MCF-10A)和4种乳腺癌细胞系(MCF-7、BT474、MDA-MB-231、HCC1937)中mRNA和蛋白表达,免疫组化染色检测FMRP在乳腺癌组织中的表达;GEO数据库分析FMRP基因在乳腺癌中的表达及与临床预后的关系;采用慢病毒感染和siRNA干扰技术分别构建FMRP过表达及干扰载体,转染人乳腺癌细胞系MCF-7,设置对照组(Control)、干扰空载体组(NC)、敲低载体组(si FMRP)以及过表达空载体组(Lv-NC)、过表达载体组(Lv-FMRP);划痕实验和Transwell实验检测细胞迁移和侵袭能力,Western blot检测各组细胞中EMT标志物[上皮标志物E-cadherin,间质标志物N-cadherin、vimentin、ZEB1(zinc finger E-box binding homeobox 1)、Slug(snail family zinc finger 2)]表达;免疫共沉淀联合质谱分析(IP-MS)验证FMRP与DEAD box RNA解旋酶-5(DEAD-box helicase5,DDX5)蛋白的互作关系,利用蛋白合成抑制剂放线菌酮(cycloheximide,CHX)、蛋白酶体抑制剂MG132检测FMRP对DDX5蛋白表达的调控作用;同时转染si DDX5载体,观察DDX5是否可以逆转FMRP过表达对细胞迁移、EMT的影响;采用免疫荧光染色检测β-catenin的定位及表达,Western blot检测Wnt/β-catenin信号通路核心标志物蛋白表达。结果FMRP在乳腺癌组织及细胞中呈高表达(P<0.05),FMRP高表达组总生存和无进展生存低于FMRP低表达组(P<0.05);敲低FMRP后MCF-7细胞迁移能力减弱,过表达FMRP促进细胞迁移(P<0.05);敲低FMRP后E-cadherin表达升高,N-cadherin、vimentin、ZEB1、Slug表达降低,抑制EMT发生,而过表达FMRP则促进EMT进程(P<0.05);FMRP与DDX5蛋白互作,且通过阻断泛素-蛋白酶体途径促进DDX5蛋白稳定性;敲低DDX5逆转FMRP过表达对细胞迁移及EMT的促进作用(P<0.05),且有Objective To investigate the role of fragile X mental retardation protein(FMRP)in promoting cell migration and epithelial-mesenchymal transition(EMT)in breast cancer(BC)and the potential mechanisms involved.Methods The mRNA and protein expressions of FMRP in MCF-10A,a normal human breast epithelial cell line,and four breast cancer cell lines,including MCF-7,BT474,MDA-MB-231,and HCC1937,were analyzed by RT-PCR and Western blot.The expression of FMRP in BC tissues was measured by immunohistochemistry(IHC).FMRP expression in BC and its relationship with clinical prognosis were analyzed using GEO database.Lentiviral infection and siRNA interference were used to construct FMRP overexpression and interference vectors,respectively,and the human breast cancer cell line MCF-7 was subsequently transfected.A Control group,an interference empty vector group(the NC group),a knockdown vector group(the siFMRP group),an overexpression empty vector group(the Lv-NC group),and an overexpression vector group(the Lv-FMRP group)were set up.The migration and invasion abilities of the cells were assessed by scratch assay and Transwell assay.The expression of EMT markers,including E-cadherin,an epithelial marker,N-cadherin,an mesenchymal markers,vimentin,zinc finger E-box binding homeobox 1(ZEB1),and snail family zinc finger 2(Slug),in the cells of each group was determined by Western blot.The interaction between FMRP and DEAD-box RNA helicase-5(DDX5)protein was analyzed by immunocoprecipitation combined with mass spectrometry(IP-MS).The regulatory effect of FMRP on DDX5 protein expression was assessed using the protein synthesis inhibitor cycloheximide(CHX)and proteasome inhibitor MG132.In addition,transfection with siDDX5 vector was conducted to observe whether DDX5 could reverse the effects of FMRP overexpression on cell migration and EMT.The localization and expression ofβ-catenin were determined by immunofluorescence staining,and the expression of core markers of Wnt/β-catenin signaling pathway was examined by Western blot.Results

关 键 词：脆性X智力障碍蛋白 DEAD box RNA解旋酶-5 上皮-间质转化 WNT/Β-CATENIN信号通路 

分 类 号：R737.9[医药卫生—肿瘤]