miRNA-200a靶向BRD4对非小细胞肺癌上皮间质转化的影响  

Effect of miRNA-200a targeting BRD4 on epithelial-mesenchymal transition in non-small cell lung cancer

作　　者：田晓静[1] 周伊兰[1] 薛宏峰[1] TIAN Xiao-Jing;ZHOU Yi-Lan;XUE Hong-Feng(Changzhou TCM Hospital Affiliated to Nanjing TCM University,Changzhou 213003,China)

机构地区：[1]南京中医药大学附属常州市中医医院检验科,常州213003

出　　处：《中国免疫学杂志》2020年第7期832-836,共5页Chinese Journal of Immunology

摘　　要：目的:探讨miRNA-200a靶向BRD4对非小细胞肺癌(NSCLC)上皮间质转化(EMT)的影响及其可能的作用途径。方法:收集2017年1月至2018年12月我院手术切除的NSCLC组织和癌旁组织标本65例,采用RT-PCR检测miRNA-200a在NSCLC组织和癌旁组织中的表达水平;构建miRNA-NC和miRNA-200a稳定细胞系,采用生物信息学和荧光素酶报告基因分析miRNA-200a和BRD4的靶向关系;pcDNA空载和pcDNA-BRD4过表达载体分别转染miRNA-NC和miRNA-200a细胞,采用Western blot检测miRNA-200a靶向BRD4对E-cadherin和Vimentin蛋白表达的影响;采用Transwell分析miRNA-200a靶向BRD4对NSCLC细胞侵袭能力的影响。结果:miRNA-200a在NSCLC组织中的相对表达水平显著低于癌旁组织,差异有统计学意义(P<0.05)。生物信息学分析表明BRD4可能是miRNA-200a的一个作用靶点;双荧光素酶报告基因结果显示,在过表达BRD4-WT的细胞中,miRNA-200a组细胞的相对荧光素酶活性显著低于miRNA-NC组细胞(P<0.05);而在过表达BRD4-MUT的细胞中,miRNA-200a组和miRNA-NC组细胞的相对荧光素酶活性无显著性差异(P>0.05)。Western blot结果显示,与miRNA-NC组比较,miRNA-200a组细胞BRD4和Vimentin的蛋白表达水平显著下降,E-cadherin的蛋白表达显著增加(P<0.05);与miRNA-200a+pcDNA组比较,miRNA-200a+BRD4组细胞中Vimentin的蛋白表达水平明显回升,E-cadherin的蛋白表达显著回落,差异有统计学意义(P<0.05)。Transwell实验显示,与miRNA-NC组比较,miRNA-200a组细胞的侵袭个数显著下降[(83.66±9.25)个比(32.73±6.02)个,P<0.05];与miRNA-200a+pcDNA组比较,miRNA-200a+BRD4组个细胞的侵袭个数明显回升,差异有统计学意义[(35.06±5.84)个比(62.02±7.28)个,P<0.05]。结论:miRNA-200a在NSCLC组织中高表达,过表达miRNA-200a可靶向下调BRD4的表达,从而抑制NSCLC中BRD4诱导的EMT和侵袭迁移。Objective:To investigate the effect of miRNA-200a targeting BRD4 on epithelial-mesenchymal transition in non-small cell lung cancer and its possible pathways.Methods:A total of 65 NSCLC tissues and adjacent tissues were collected from January 2017 to December 2018.The mRNA expression of miRNA-200a in NSCLC tissues and adjacent tissues were detected by RT-PCR.miRNA-NC and miRNA-200a stable cell lines were constructed,and bioinformatics and dual-luciferase reporter gene assay were used to analyze the targeting relationship between miRNA-200a and BRD4.miRNA-NC and miRNA-200a cells were transfected with pcDNA empty and pcDNA-BRD4 overexpression vectors,respectively.The effect of miRNA-200a targeting BRD4 on the expression of E-cadherin and Vimentin was detected by Western blot.Transwell was used to analyze the effect of miRNA-200a targeting BRD4 on the invasion ability of NSCLC cells.Results:The relative expression of miRNA-200a in NSCLC tissues was significantly lower than that in adjacent tissues(P<0.05).Bioinformatics indicated that BRD4 may be a target of miRNA-200a.The dual-luciferase reporter gene assay showed that the relative luciferase activity of miRNA-200a was significantly lower than that of miRNA-NC in cells overexpressing BRD4-WT(P<0.05),while in the cells overexpressing BRD4-MUT,there was no significant difference in relative luciferase activity between miRNA-200a and miRNA-NC(P>0.05).Western blot showed that compared with miRNA-NC group,the protein expression of BRD4 and Vimentin in miRNA-200a group decreased significantly,and the expression of E-cadherin increased significantly(P<0.05).Compared with the miRNA-200a+pcDNA group,the expression of Vimentin in the miRNA-200a+BRD4 group was significantly increased,and the expression of E-cadherin was significantly decreased(P<0.05).Transwell showed that compared with the miRNA-NC group,the number of invasion in the miRNA-200a group was significantly decreased[(83.66±9.25)vs(32.73±6.02),P<0.05].Compared with miRNA-200a+pcDNA group,the number of invasion i

关 键 词：miRNA-200a BRD4 非小细胞肺癌 上皮间质转化 侵袭 

分 类 号：R734[医药卫生—肿瘤]