异黏蛋白调控三阴性乳腺癌细胞干性的机制研究  

Role of metadherin in regulating stemness of triple negative breast cancer cells and its mechanism

作　　者：乌帆 陈国华[1] 张良 王耕[1] 李文仿[1] Wu Fan;Chen Guohua;Zhang Liang;Wang Geng;Li Wenfang(Breast Disease Center,Taihe Hospital Affiliated to Hubei Medical College,Shiyan 442000,Hubei Province,China;School of Clinical Medicine,Jinzhou Medical University,Jinzhou 121001,China)

机构地区：[1]湖北医药学院附属太和医院乳腺中心,湖北十堰442000 [2]锦州医科大学临床医学院,121001

出　　处：《中华乳腺病杂志（电子版）》2020年第4期221-227,共7页Chinese Journal of Breast Disease(Electronic Edition)

基　　金：湖北省十堰市科技局指导项目(17Y17);湖北省教育厅指导项目(B2019109)。

摘　　要：目的研究异黏蛋白(MTDH)对三阴性乳腺癌干性的调控机制,并探讨miR-30a-5p对MTDH的调控作用。方法(1)回顾性分析2017年5月至2018年9月湖北医药学院附属太和医院手术治疗的37例三阴性乳腺癌患者的肿瘤组织标本及同期非三阴性乳腺癌手术切除标本37例。采用免疫组织化学方法检测所有标本中MTDH的表达。(2)用定量RT-PCR和Western blot检测MCF-10A、MCF-7、MDA-MB-231、MDA-MB-4684种细胞系中MTDH的mRNA和蛋白表达。(3)为了探讨MTDH对三阴性乳腺癌细胞干性、细胞增殖及Wnt/β-catenin信号通路的影响,用MTDH的siRNA转染三阴性乳腺癌细胞系MDA-MB-231和MDA-MB-468(siMTDH组),无效干扰RNA转染的细胞作为对照组。用Western blot检测MDA-MB-231、MDA-MB-468细胞系中干细胞标志物sox2、Nanog及CD133相对表达量,用细胞克隆实验观察细胞克隆数,用Western blot检测Wnt/β-catenin信号通路蛋白β-catenin、Myc和cyclin D1的表达。(4)荧光素酶报告实验:为评估miR-30a-5p能否靶向调控MTDH,用MTDH的3′-UTR和miRNA-30a-5p合成含有MTDH 3′-UTR野生型和突变型荧光素酶报告质粒,分别转染HepG2细胞,pGL3空载体转染细胞作为空载体组,24 h后观察荧光。三阴性乳腺癌与非三阴性乳腺癌组织中MTDH蛋白表达比较用t检验。4种细胞系中MTDH mRNA及蛋白表达比较用方差分析。对照组与siMTDH组中sox2、Nanog、CD133、β-catenin、Myc、cyclin D1蛋白表达及细胞克隆数比较用t检验。细胞相对荧光比值比较采用方差分析。组间两两比较采用LSD法。结果(1)三阴性乳腺癌组织中MTDH蛋白相对表达量为2.82±1.37,而非三阴性乳腺癌组织为5.65±2.02,差异有统计学意义(t=-7.046,P<0.001)。(2)在4种细胞系(MCF-7、MDA-MB-231、MDA-MB-468及MCF-10A)中,MTDH mRNA表达比较,差异有统计学意义(F=7155.320,P<0.001)。两两比较结果显示:与正常乳腺细胞(MCF-10A)比较,乳腺癌细胞(MCF-7、MDA-MB-231、MDA-MB-468)中MTDH mRNA表达�Objective To investigate the role of metadherin(MTDH)in regulating stem cell characteristics of triple negative breast cancer and its mechanism and explore the regulation of MTDH by miR-30a-5p.Methods(1)The tumor tissue specimens of 37 triple-negative breast cancer(TNBC)patients who underwent surgery and 37 non-TNBC tissue specimens in Taihe Hospital Affiliated to Hubei Medical College from May 2017 to September 2018 were collected for a retrospective study.Immunohistochemistry was used to detect the expression of MTDH in all specimens.(2)The mRNA and protein expression of MTDH in four cell lines(MCF-10A,MCF-7,MDA-MB-231 and MDA-MB-468)by quantitative RT-PCR and Western blot.(3)To explore the effect of MTDH on the stemness and proliferation of TNBC cells and the Wnt/β-catenin signaling pathway,TNBC cell lines MDA-MB-231 and MDA-MB-468 were transfected with MTDH siRNA(siMTDH group),and the cells transfected with invalid interfering RNA served as the control group.The relative expression of stem cell markers sox2,Nanog and CD133 was determined in MDA-MB-231 and MDA-MB-468 cell lines by Western blot.Cell cloning experiments were used to observe the number of cell clones and Western blot was used to detect the expression of the Wnt/β-catenin signaling pathway proteins(β-catenin,Myc and cyclin D1).(4)To investigate the targeted regulation of MTDH by miRNA-30a-5p,the luciferase reporter assay,3′-UTR of MTDH and miRNA-30a-5p were used to synthesize wild-type and mutant luciferase reporter plasmids containing 3′-UTR of MTDH,and then the HepG2 cells were transfected with wild-type and mutant plasmids,respectively.The HepG2 cells transfected with pGL3 empty vector served as a blank control group.The fluorescence of cells was observed 24 h later.The protein expression of MTDH was compared between TNBC tissues and non-TNBC tissues using t test.The mRNA and protein expression of MTDH was compared among four cell lines using analysis of variance.The protein expression of sox2,Nanog,CD133,β-catenin,Myc,cyclin D1 and the

关 键 词：乳腺肿瘤 干细胞 微RNAS 异黏蛋白 

分 类 号：R737.9[医药卫生—肿瘤]