microRNA-130b促进三阴性乳腺癌细胞及其作用机制  

microRNA-130b promotes triple-negative breast cancer cell and mechanism

作　　者：金薇[1] 罗祎[1] 刘溦薇[1] 陈国庆[1] 周鸣[1] 

机构地区：[1]上海交通大学医学院附属同仁医院普外科,上海200336

出　　处：《外科理论与实践》2017年第1期49-56,共8页Journal of Surgery Concepts & Practice

摘　　要：目的:探讨microRNA-130b(miR-130b)的表达差异对三阴性乳腺癌(triple-negative breast cancer,TNBC)MDA-MB-231细胞增殖、迁移及侵袭能力的影响及机制。方法 :通过Lipofectamine TM 2000将miR-130b抑制剂或模拟物及阴性对照转染到MDA-MB-231细胞中。应用实时定量聚合酶链反应(quantitative real time-polymerase chain reaction,qRT-PCR)检测miR-130b表达和转染效率。应用MTT实验、克隆形成实验、划痕实验以及transwell实验,检测miR-130b模拟物或抑制剂对MDA-MB-231细胞功能影响。应用生物信息学网站寻找miR-130b可能的靶基因。应用双荧光素酶载体实验验证预测的靶基因,并通过qRT-PCR以及Western印迹实验进一步证实。结果:MDA-MB-231细胞转染miR-130b抑制剂或模拟物。与阴性对照组相比,模拟物组miR-130b的表达显著升高,抑制剂组miR-130b的表达显著下降,差异有统计学意义(P<0.01)。与阴性对照组相比,模拟物组细胞增殖(P<0.05)、克隆形成(P<0.05)、侵袭(P<0.01)及迁移(划痕试验P<0.05,transwell试验P<0.05)能力明显增高。相反,抑制剂组细胞增殖(P<0.05)、克隆形成(P<0.05)、侵袭及迁移(划痕试验P<0.05,transwell试验P<0.01)能力较阴性对照组明显受抑制。生物信息学技术预测CYLD为miR-130b的潜在靶基因。双荧光素酶载体实验结果显示转染miR-130b模拟物+psciCHECK2-CYLD 3′UTR(野生型)后荧光素酶活性较转染阴性对照+psciCHECK2-CYLD 3′UTR(野生型)降低57.21%(P<0.001)。此外,与阴性对照组相比,CYLD基因m RNA水平在抑制剂组或模拟物组均无明显改变(P>0.05);但CYLD蛋白表达在抑制剂组明显上调,模拟物组的表达明显下降(P<0.001)。结论:miR-130b可促进TNBC MDA-MB-231细胞的增殖、侵袭及迁移,其作用至少部分通过抑制CYLD基因实现。Objective To explore the effect of microRNA-130b（miR-130b） expression on proliferation, invasion and migration of triple-negative breast cancer（TNBC） cell and the mechanism. Methods TNBC cell lines MDA-MB-231 were transiently transfected with miR-130b inhibitor, mimic or the negative control（NC） by LipofectamineTM2000. Successful transfection was confirmed by quantitative real time polymerase chain reaction（qRT-PCR）. The function of MDA-MB-231 cells was investigated by the assays of MTT, colony formation, scratch and transwell test after transfection with miR-130b inhibitor mimic and NC. The bioinformatics software was used to look for the potential target gene of miR-130b. Dual-luciferase reporter gene assay was used to verify whether miR-130b was bound to 3′untranslated regions（3′UTR） of potential target gene and the effect was further verified by qRT-PCR and Western blot analysis. Results MDA-MB-231 cells were transfected with miR-130b inhibitor, mimic and NC. The expression of miR-130b increased significantly in mimic group and decreased in inhibitor group when compared with those in NC group with statistical difference significantly（P〈0.01）.The promotion of cell proliferation（P〈0.05）, colony formation（P〈0.05）, invasion（P〈0.01） and migration（scratch assay P〈0.05, transwell test P〈0.05） was promoted in mimic group, whereas down-regulated expression of miR-130b suppressed the ability of proliferation（P〈0.05）, colony formation（P〈0.05）, migration（scratch assay P〈0.05, transwell test P〈0.01） and invasion（P〈0.01） when compared with the control cells. According to prediction, CYLD is a potential target of miR-130b based on the prediction with bioinformatics software. Dual-luciferase reporter gene assay revealed that luciferase activity was down-regulated 57.21% in mimic group of transfection with miR-130b mimic ＋psciCHECK2-CYLD 3′ UTR（wild）compared with that in negative group of transfection with miR-NC＋psciCHECK2- CYLD 3′U

关 键 词：miR-130b CYLD 三阴性乳腺癌 增殖 侵袭 

分 类 号：R737.9[医药卫生—肿瘤]