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

Q3 Medicine
Jia Cao, Jing Wang, Bin Shi, Xiaolan Ma, Weichao Wu, Nan Wang
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引用次数: 0

Abstract

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 of β-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 ubiquitin-proteasome pathway. DDX5 knockdown reversed the effect of FMRP overexpression to promote cell migration and EMT (P<0.05), effectively inhibited β-catenin nuclear translocation, and decreased β-catenin nuclear distribution. Furthermore, it was found that the expression of p-β-catenin, GSK3β and 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.

[DDX5靶向脆性X智力迟钝蛋白调控Wnt/β-catenin信号通路,促进乳腺癌的上皮间充质转化】。]
目的研究脆性X智障蛋白(FMRP)在促进乳腺癌(BC)细胞迁移和上皮-间质转化(EMT)中的作用及其潜在机制:方法:采用RT-PCR和Western blot技术分析了FMRP在正常人乳腺上皮细胞MCF-10A以及MCF-7、BT474、MDA-MB-231和HCC1937等四种乳腺癌细胞系中的mRNA和蛋白表达。免疫组化法(IHC)检测了FMRP在乳腺癌组织中的表达。利用GEO数据库分析了FMRP在BC中的表达及其与临床预后的关系。分别用慢病毒感染和 siRNA 干扰构建 FMRP 过表达载体和干扰载体,然后转染人乳腺癌细胞系 MCF-7。实验分为对照组、干扰空载体组(NC 组)、敲除载体组(siFMRP 组)、过表达空载体组(Lv-NC 组)和过表达载体组(Lv-FMRP 组)。通过划痕试验和 Transwell 试验评估细胞的迁移和侵袭能力。各组细胞中EMT标记物(包括上皮标记物E-cadherin、间充质标记物N-cadherin、波形蛋白、锌指E盒结合同工酶1(ZEB1)和蜗牛家族锌指2(Slug))的表达均通过Western印迹法测定。免疫共沉淀结合质谱法(IP-MS)分析了FMRP与DEAD-box RNA螺旋酶-5(DDX5)蛋白之间的相互作用。使用蛋白质合成抑制剂环己亚胺(CHX)和蛋白酶体抑制剂 MG132 评估了 FMRP 对 DDX5 蛋白表达的调控作用。此外,还用 siDDX5 载体转染观察 DDX5 是否能逆转 FMRP 过表达对细胞迁移和 EMT 的影响。免疫荧光染色检测了β-catenin的定位和表达,Western blot检测了Wnt/β-catenin信号通路核心标记物的表达:FMRP在BC组织和细胞中高表达(PFMRP高表达组明显低于FMRP低表达组),过表达FMRP促进细胞迁移(PFMRP敲除后,E-cadherin的表达增加,而N-cadherin、vimentin、ZEB1和Slug的表达水平降低,抑制了EMT的发生),而过表达FMRP促进细胞迁移(PFMRP敲除后,E-cadherin的表达增加,而N-cadherin、vimentin、ZEB1和Slug的表达水平降低,抑制了EMT的发生)。相反,FMRP的过表达促进了EMT过程(PDDX5敲除逆转了FMRP过表达促进细胞迁移和EMT的效应(PDDX5下调(PFMRP过表达(PConclusion:FMRP靶向DDX5,通过激活Wnt/β-catenin信号通路促进BC细胞迁移和EMT。
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来源期刊
四川大学学报(医学版)
四川大学学报(医学版) Biochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
0.70
自引率
0.00%
发文量
8695
期刊介绍: "Journal of Sichuan University (Medical Edition)" is a comprehensive medical academic journal sponsored by Sichuan University, a higher education institution directly under the Ministry of Education of the People's Republic of China. It was founded in 1959 and was originally named "Journal of Sichuan Medical College". In 1986, it was renamed "Journal of West China University of Medical Sciences". In 2003, it was renamed "Journal of Sichuan University (Medical Edition)" (bimonthly). "Journal of Sichuan University (Medical Edition)" is a Chinese core journal and a Chinese authoritative academic journal (RCCSE). It is included in the retrieval systems such as China Science and Technology Papers and Citation Database (CSTPCD), China Science Citation Database (CSCD) (core version), Peking University Library's "Overview of Chinese Core Journals", the U.S. "Index Medica" (IM/Medline), the U.S. "PubMed Central" (PMC), the U.S. "Biological Abstracts" (BA), the U.S. "Chemical Abstracts" (CA), the U.S. EBSCO, the Netherlands "Abstracts and Citation Database" (Scopus), the Japan Science and Technology Agency Database (JST), the Russian "Abstract Magazine", the Chinese Biomedical Literature CD-ROM Database (CBMdisc), the Chinese Biomedical Periodical Literature Database (CMCC), the China Academic Journal Network Full-text Database (CNKI), the Chinese Academic Journal (CD-ROM Edition), and the Wanfang Data-Digital Journal Group.
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