微RNA-378a-3p靶向SUFU基因促进乳腺癌细胞的增殖和迁移  

Micro RNA-378a-3p targeting SUFU gene promotes proliferation and migration of breast cancer cells

作　　者：王嘉琳 高维强[1,2] 方煜翔 WANG Jialin;GAO Weiqiang;FANG Yuxiang(Renji-Med X Clinical Stem Cell Research Center,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China;School of Biomedical Engineering&Med-X Research Institute,Shanghai Jiao Tong University,Shanghai 200030,China)

机构地区：[1]上海交通大学医学院附属仁济医院临床干细胞研究中心,上海200127 [2]上海交通大学生物医学工程学院Med-X研究院,上海200030

出　　处：《肿瘤》2021年第5期325-340,共16页Tumor

基　　金：国家自然科学基金(编号:81874097;81872406)。

摘　　要：目的:研究微RNA(microRNA,miRNA,miR)-378a-3p对乳腺癌细胞体外增殖、凋亡和迁移能力的影响,以及可能的作用机制。方法:利用GEO(Gene Expression Omnibus)数据库分析乳腺癌miR-378a-3p高表达患者与低表达患者的生存情况。使用实时荧光定量PCR法检测人乳腺癌细胞系以及正常人乳腺上皮细胞中miR-378a-3p的表达水平。采用慢病毒感染的方法将携带miR-378a-3p-模拟物(miR-378a-3p-mimics)的重组慢病毒转入人乳腺癌MCF-7细胞,构建稳定过表达miR-378a-3p的MCF-7细胞;将携带miR-378a-3p-抑制子(miR-378a-3p-inhibitor)的重组慢病毒转入人乳腺癌MDA-MB-231细胞,构建稳定抑制miR-378a-3p表达的MDA-MB-231细胞。采用CCK-8法、Transwell小室法和FCM法检测过表达miR-378a-3p或抑制其表达对乳腺癌细胞增殖、凋亡和迁移的影响。通过生物信息学分析以及双荧光素酶报告系统验证miR-378a-3p对其靶基因SUFU(Hedgehog信号通路负调控因子)的直接靶向调控作用。miR-378a-3p表达水平改变后,通过免疫共沉淀法验证SUFU蛋白与人GLI家族锌指蛋白1(GLI family zinc finger 1,GLI1)之间的相互作用,并采用蛋白质印迹法检测2者在细胞核和细胞质中表达水平的变化情况。在过表达miR-378a-3p或抑制其表达的基础上,使用Hedgehog信号通路抑制剂或激活剂处理乳腺癌细胞,然后采用CCK-8法、Transwell小室法和FCM法检测过表达miR-378a-3p或抑制其表达是否通过激活或抑制Hedgehog信号通路调控乳腺癌细胞的增殖、凋亡和迁移。结果:高表达miR-378a的乳腺癌患者的预后较低表达miR-378a的乳腺癌患者的差。miR-378a-3p在多种乳腺癌细胞中的表达水平均明显上调(P值均<0.001)。乳腺癌MCF-7细胞稳定过表达miR-378a-3p后,MCF-7细胞的体外增殖(P<0.05)和抗凋亡(P<0.01)能力均明显增强;反之,在乳腺癌MDA-MB-231细胞中沉默miR-378a-3p表达后,MDA-MB-231细胞的体外增殖(P<0.000 1)、抗凋亡(P<0.05)和迁移(P<0.0Objective:To investigate the effects of microRNA (microRNA,miRNA,miR)-378a-3p on the proliferation,apoptosis and migration of breast cancer cells in vitro,and the possible mechanisms.Methods:The GEO (Gene Expression Omnibus) database was used to analyze the survival of breast cancer patients with high or low expression of miR-378a-3p.The real-time fluorescent quantitative PCR was performed to detect the expression level of miR-378a-3p in human breast cancer cell lines and normal human breast epithelial cells.A recombinant lentivirus carrying miR-378a-3p-mimics was transfected into human breast cancer MCF-7 cells using a lentiviral infection method to construct MCF-7 cells that stably overexpress miR-378a-3p,and a recombinant lentivirus carrying miR-378a-3p-inhibitor was transferred into human breast cancer MDA-MB-231 cells to construct MDA-MB-231 cells that stably inhibit miR-378a-3p expression.The effects of overexpression of miR-378a-3p or inhibition of its expression on proliferation,apoptosis and migration of breast cancer cells were examined by CCK-8 assay,Transwell assay and FCM.The direct targeting and regulation of miR-378a-3p on its target gene SUFU—a negative regulator of Hedgehog signaling pathway,was verified by bioinformatics analysis and dual luciferase reporter system.After the altered expression of miR-378a-3p,the interaction between SUFU protein and human GLI family zinc finger 1 (GLI1) protein was verified by immunoprecipitation,and the altered expression levels of both proteins in the nucleus and cytoplasm were detected by Western blotting.On the basis of overexpression of miR-378a-3p or inhibition of its expression,breast cancer cells were treated with Hedgehog signaling pathway inhibitor or activator,followed by CCK-8 assay,Transwell assay and FCM to detect whether the overexpression of miR-378a-3p or inhibition of its expression regulated the proliferation,apoptosis and migration of breast cancer cells through activation or inhibition of Hedgehog signaling pathway.Results:The prognosis of

关 键 词：乳腺肿瘤 微RNAS 细胞增殖 细胞凋亡 细胞运动 HEDGEHOG信号通路 

分 类 号：R737.9[医药卫生—肿瘤]