摘要
目的:探讨上调或下调微RNA(microRNA,miRNA,miR)-195-5p对人三阴性乳腺癌MDA-MB-231和BT-549细胞增殖、迁移和侵袭的影响,及可能的作用机制。方法:应用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库分析miR-195-5p在乳腺癌中的表达水平。将miR-195-5p-模拟物(mimics)转入MDA-MB-231细胞构建外源性miR-195-5p高表达的重组细胞,同时将miR-195-5p-抑制剂(inhibitor)转入BT-549细胞构建沉默内源性miR-195-5p表达的重组细胞。实时荧光定量PCR法检测重组细胞中miR-195-5p的表达水平;MTT法检测细胞的增殖能力;划痕愈合实验及Transwell小室法分别检测细胞的横向、纵向迁移能力及侵袭能力;实时荧光定量PCR法检测上皮-间质转化(epithelial-mesenchymal transition,EMT)标志物E-钙黏蛋白(E-cadherin)、转录因子Snail和波形蛋白(vimentin)及迁移相关因子yes相关蛋白1(yes-associated protein-1,YAP-1)mRNA的表达水平;蛋白质印迹法检测EMT相关标志物锌指蛋白E盒结合同源异形盒1(zinc finger E-box binding homeobox 1,ZEB1)、E-cadherin、N-钙黏蛋白(N-cadherin)和vimentin以及迁移相关标志物YAP-1和磷酸化YAP-1蛋白的表达水平。结果:miR-195-5p在乳腺癌中的表达水平低。MDA-MB-231/mimics细胞中miR-195-5p的表达水平明显上调(P<0.001),BT-549/inhibitor细胞中miR-195-5p的表达水平显著下调(P<0.001)。miR-195-5p表达上调后MDA-MB-231/mimics细胞的增殖能力明显减弱(P<0.001),miR-195-5p表达下调后BT-549/inhibitor细胞的增殖能力明显增强(P<0.05);MDA-MB-231/mimks细胞的横向和纵向迁移及侵袭能力均被明显抑制(P值均<0.05),BT-549/inhibitor细胞的横向和及纵向迁移能力及侵袭能力均被明显提高(P值均<0.05)。实时荧光定量PCR法显示,MDAMB-231/mimics细胞中上皮细胞标志物E-cadherin mRNA表达水平明显升高(P<0.05),而间质细胞标志物vimentin和N-cadherin mRNA的表达水平则显著下调(P<0.05);蛋白质印迹法检测结果显示,MDAMB-231/mimics细胞中E-cadherin蛋白表达水平明显升高(P<0.001),而vimentin(P<0.05)、N-cadherin(P<0.01)和ZEB1(P<0.05)蛋白的表达水平则明显降低,提示EMT过程可能被抑制;MDA-MB-231/mimics细胞中迁移相关标志物YAP-1及其磷酸化蛋白的表达水平均明显下调(P<0.05和P<0.01)。结论:miR-195-5p能抑制乳腺癌细胞的增殖、侵袭、迁移和EMT的发生,该过程可能与HippoYAP信号通路的抑制相关。
Objective:To investigate the effects of up-or down-regulated microRNA(miRNA,miR)-195-5p on the growth,migration and invasion of human triple negative breast cancer MDAMB-231 and BT-549 cells,and to explore the potential mechanism.Methods:The Cancer Genome Atlas(TCGA)database was used to analyze the expression level of miR-195-5p in breast cancer.MiR-195-5p-mimics was transfected into MDA-MB-231 cells to construct the recombinant cells with exogenous miR-195-5p overexpression.MiR-195-5p-inhibitor was transfected into BT-549 cells to construct the recombinant cells with endogenous miR-195-5p gene silence.The expression level of miR-195-5p in the recombinant cells was detected by realtime fluorescent quantitative PCR.The proliferation of MDA-MB-231 and BT-549 cells was detected by MTT assay,the longitudinal migration and invasion abilities were detected by Transwell migration and invasion assays,the lateral migration ability was evaluated by wound healing assay.The mRNA expression levels of migration-associated factor yes-associated protein-1(YAP-1),and epithelialmesenchymal transition(EMT)markers(including E-cadherin,snail and vimentin)were detected by real-time fluorescent quantitative PCR.Meantime,the expression levels of EMT markers(including zinc finger E-box binding homeobox 1(ZEB1),E-cadherin,N-cadherin and vimentin),YAP-1,and phosphorylated YAP-1(p-YAP-1)were detected by Western blotting.Results:The expression of miR-195-5p in breast cancer tissues was low.The expression of miR-195-5p in MDA-MB-231 cells was lower than that in BT-549 cells(P<0.001).After the transfection of miR-195-5p-mimics into MDA-MB-231 cells,the expression of miR-195-5p was significantly increased(P<0.001);while the expression of miR-195-5p in BT-549 cells transfected with miR-195-5p-inhibitor was decreased(P<0.001).After the over-expression of miR-195-5p,the proliferation,migration and invasion abilities of MDA-MB-231 cells were decreased(all P<0.05);but the opposite phenomenon was observed in BT-549 cells with down-regulation of miR-195-5p(all P<0.05).The expression level of E-cadherin mRNA increased,but the expression levels of YAP-1,vimentin and snail mRNAs decreased in MDA-MB-231 cells transfected with miR-195-5p-mimics(all P<0.05).The expression level of E-cadherin protein increased(P<0.001),but the expression levels of ZEB1(P<0.05),N-cadherin(P<0.01)and vimentin proteins(P<0.05)decreased in MDAMB-231 cells with miR-195-5p overexpression,suggesting that the EMT process might be inhibited.The expression levels of migration-related markers YAP-1 and p-YAP-1 proteins in MDA-MB-231 cells with miR-195-5p overexpression were significantly down-regulated(P<0.05 and P<0.01).Conclusion:The expression of miR-195-5p is down-regulated in breast cancer,and can inhibit the proliferation,migration,invasion and EMT of breast cancer cells,which may be related to reducing the activity of Hippo/YAP signaling pathway.
作者
孙恃雷
唐敏
万群
孙子久
胡静
方玉婷
张彦
SUN Shilei;TANG Min;WAN Qun;SUN Zijiu;HU Jing;FANG Yuting;ZHANG Yan(Key Laboratory of Clinical Laboratory Diagnosis,Ministry of Education,College of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2020年第2期122-132,共11页
Tumor
基金
国家自然科学基金资助项目(编号:81172017,81974449).
作者简介
Correspondence to:张彦,E-mail:yanzhang@cqmu.edu.cn.
