miR-200a-3p在胆囊癌中的表达及其作用与机制  

Expression of miR-200a-3p in gallbladder cancer and its effects and mechanism of action

作　　者：孔祥海 胡敏[1] 王笛乐[1] 王李理 何涛[1] KONG Xianghai;HU Min;WANG Dile;WANG Lili;HE Tao(Department of General Surgery,the Central Hospital of Wuhan,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430014,China)

机构地区：[1]华中科技大学同济医学院附属武汉中心医院普通外科,湖北武汉430014

出　　处：《中国普通外科杂志》2021年第8期917-925,共9页China Journal of General Surgery

摘　　要：背景与目的:miR-200a-3p参与了多种肿瘤的生物学过程的调控,但在不同肿瘤中起着不同的作用(促癌基因或抑癌基因)。目前,其在胆囊癌中的作用尚不清楚。因此,本研究探讨miR-200a-3p在胆囊癌中的表达及其对胆囊癌细胞生物学功能的影响与机制。方法:采用qRT-PCR法测定32例胆囊癌及癌旁组织、胆囊癌细胞系(GBC-SD、SGC-996、NOZ)及正常人胆囊上皮细胞系HGBEC中miR-200a-3p的表达。采用LipofectamineTM3000试剂盒将GBC-SD及NOZ细胞系分别转染miR-200a-3p模拟物(miR-200a-3p过表达组)、miR-200a-3p抑制物序列(miR-200a-3p沉默组)及阴性对照序列(阴性对照组)。MTT实验测定细胞增殖能力,Transwell实验测定细胞侵袭能力,MiRBD/Targetscan7.2/starBase2.0/miRtarbase网站预测miR-200a-3p的下游靶基因,并采用荧光素酶实验验证。Western blot检测上述3组细胞中靶基因与上皮间质转化(EMT)相关分子蛋白(E-cadherin、vimentin)的表达。结果:q RT-PCR结果显示,胆囊癌组织中miR-200a-3p表达量低于癌旁组织,所有胆囊癌细胞系中miR-200a-3p表达量低于正常人胆囊上皮系HGBEC(均P<0.05)。GBC-SD及NOZ细胞系转染后,与各自的阴性对照组比较,两种细胞的miR-200a-3p过表达组miR-200a-3p表达量明显升高、增殖与侵袭能力明显减弱,两种细胞的miR-200a-3p沉默组miR-200a-3p表达量明显降低、增殖与侵袭能力明显增强(均P<0.05)。在线网站预测显示,miR-200a-3p和Notch2存在潜在结合位点;荧光素酶实验显示,miR-200a-3p导致Notch2野生型质粒pmirGLO-Notch2-3’UT WT荧光素酶活性明显降低,而miR-200a-3p对Notch2突变型质粒pmirGLO-Notch2-3’UTR MUT的荧光素酶活性没有影响。Western blot结果显示,两种细胞的miR-200a-3p过表达组与各自的阴性对照组比较,E-cadherin蛋白表达量升高、vimentin蛋白与Notch2蛋白表达量降低,而3种蛋白在两种细胞的miR-200a-3p沉默组则呈相反的变化(均P<0.05)。结论:miR-200a-3p�Background and Aims:MiR-200 a-3 p participates in the regulation of biological processes in a variety of tumors,but it exerts different effects in different tumors(oncogene or tumor suppressor).At present,its role in gallbladder cancer is still unclear.Therefore,this study was conducted to investigate the expression of miR-200 a-3 p in gallbladder cancer as well as its effects on biological behaviors of gallbladder cancer cells and the mechanism.Methods:The expressions of miR-200 a-3 p in 32 specimens of gallbladder cancer tissue and adjacent tissue as well as in different gallbladder cancer cell lines(GBC-SD,SGC-996,and NOZ)and normal human gallbladder epithelial cell line HGBEC were determined by qRT-PCR method.The GBC-SD and NOZ cell lines were transfected with miR-200 a-3 p-mimics(miR-200 a-3 p overexpression group),miR-200 a-3 p-inhibitors(miR-200 a-3 p silencing group)and scramble sequences(negative control group)respectively using LipofectamineTM 3000 kit.The proliferation and invasion abilities of the cells were examined by MTT assay and Transwell assay.The downstream target genes of miR-200 a-3 p were predicted using MiRBD/Targetscan7.2/starBase2.0/miRtarbase website,and were verified by Luciferase experiment.The protein expressions of the target genes and the molecules(E-cadherin and vimentin)associated with epithelial-to-mesenchymal transition(EMT)in above three groups of cells were measured by Western blot analysis.Results:The results of qRT-PCR showed that the expression level of miR-200 a-3 p in gallbladder cancer tissue was lower than that in adjacent tissue,and the expression levels of miR-200 a-3 p in all gallbladder cancer cell lines were lower than that in normal human gallbladder epithelial cell line HGBEC(all P<0.05).In GBC-SD and NOZ cell lines after transfection,the expressions of miR-200 a-3 p were significantly increased and the proliferative and invasion abilities were significantly decreased in miR-200 a-3 p overexpression groups of both cell lines,while the expressions of miR-200 a-3 p

关 键 词：胆囊肿瘤 微RNAS 细胞增殖 肿瘤侵润 上皮-间质转化 

分 类 号：R735.8[医药卫生—肿瘤]