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

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),且有效抑制β-catenin核转位,降低β-catenin核分布;进一步发现DDX5下调后p-β-catenin、GSK3β、Axin2蛋白表达升高,C-myc蛋白表达降低(P<0.05),而联合FMRP过表达逆转上述蛋白的表达(P<0.05)。结论FMRP靶向DDX5通过激活Wnt/β-catenin信号通路促进乳腺癌细胞迁移和EMT进程。

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 FMRP was highly expressed in BC tissues and cells(P<0.05),and overall survival(OS)and recurrence-free survival(RFS)of the FMRP high expression group were significantly lower than those of the FMRP low expression group(P<0.05).The migration ability of MCF-7 cells was weakened after FMRP knockdown,while overexpression of FMRP promoted cell migration(P<0.05).After FMRP knockdown,the expression of E-cadherin was increased,while the expression levels of N-cadherin,vimentin,ZEB1,and Slug were decreased,which inhibited the occurrence of EMT.In contrast,the overexpression of FMRP promoted the EMT process(P<0.05).FMRP interacted with DDX5 protein and promoted DDX5 protein stability by blocking the ubiquitinproteasome pathway.DDX5 knockdown reversed the effect of FMRP overexpression to promote cell migration and EMT(P<0.05),effectively inhibitedβ-catenin nuclear translocation,and decreasedβ-catenin nuclear distribution.Furthermore,it was found that the expression of p-β-catenin,GSK3βand Axin2 protein was increased and the expression of C-myc protein was decreased after DDX5 downregulation(P<0.05).On the other hand,the expression of these proteins was reversed by combined FMRP overexpression(P<0.05).Conclusion FMRP targets DDX5 and promotes BC cell migration and EMT via the activation of the Wnt/β-catenin signaling pathway.