长链非编码RNA-H19特异性吸附微RNA-760调控nanog基因表达促进非小细胞肺癌细胞增殖和迁移  

Long non-coding RNA-H19 promotes proliferation and migration of non-small cell lung cancer cells through sponging microRNA-760 to regulate nanog gene expression

作　　者：李莎[1] 朱怡卿 石荟 戈霞晖 许靖[3] 商艳[1,3] 王芳 白冲 LI Sha;ZHU Yi-qing;SHI Hui;GE Xia-hui;XU Jing;SHANG Yan;WANG Fang;BAI Chong(Department of Respiratory and Critical Care Medicine,Changhai Hospital,Naval Medical University(Second Military Medical University),Shanghai 200433,China;Department of Medical Genetics,College of Basic Medical Sciences,Naval Medical University(Second Military Medical University),Shanghai 200433,China;Department of Respiratory Medicine,Seventh People’s Hospital of Shanghai,Shanghai University of Traditional Chinese Medicine,Shanghai 200137,China)

机构地区：[1]海军军医大学(第二军医大学)长海医院呼吸与危重症医学科,上海200433 [2]海军军医大学(第二军医大学)基础医学院医学遗传学教研室,上海200433 [3]上海中医药大学附属第七人民医院呼吸科,上海200137

出　　处：《第二军医大学学报》2019年第8期854-859,共6页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(81570020,81300018);浙江省公益技术应用研究计划项目(2016C33216);浦东新区科技发展基金(PKJ2016-Y49)~~

摘　　要：目的探讨长链非编码RNA(lncRNA)-H19对非小细胞肺癌(NSCLC)细胞增殖、迁移的影响及分子机制。方法收集2015年10月至2016年5月海军军医大学(第二军医大学)长海医院确诊的20例NSCLC患者手术切除的NSCLC组织及相应的癌旁正常组织。通过实时荧光定量PCR(qPCR)检测NSCLC组织及癌旁正常组织,人NSCLC细胞系A549、NCI-H1299和人正常肺上皮细胞系BEAS-2B中lncRNA-H19的表达。在A549细胞中过表达lncRNA-H19后,采用CCK-8法和Transwell实验分别检测细胞的增殖和迁移能力。利用生物信息学分析预测lncRNA-H19与微RNA(miRNA)-760的结合位点,通过双荧光素酶报告基因实验分析lncRNA-H19对miRNA-760的吸附作用,采用qPCR和蛋白质印迹法分别检测lncRNA-H19过表达对miRNA-760及下游靶基因nanog表达的影响。通过对A549细胞转染miRNA-760模拟剂过表达miRNA-760后,检测过表达miRNA-760对lncRNA-H19促进NSCLC细胞增殖和迁移作用的影响。结果LncRNA-H19在NSCLC组织和A549、NCI-H1299细胞中均呈高表达,分别与其在癌旁正常组织和BEAS-2B细胞中的表达水平相比差异均有统计学意义(P均<0.01)。与对照组相比,过表达lncRNA-H19后A549细胞的增殖能力增强(P<0.05)、迁移能力提高(P<0.01)。在线数据库starBase v3.0预测分析结果显示lncRNA-H19能特异性吸附miRNA-760。双荧光素酶报告基因检测结果显示lncRNA-H19与miRNA-760之间存在特异性结合。与对照组相比,过表达lncRNA-H19可抑制A549细胞中miRNA-760的表达(P均<0.05),并促进其下游基因nanog在mRNA和蛋白水平的表达(P均<0.01)。过表达miRNA-760可抑制lncRNA-H19对A549细胞增殖和迁移的促进作用,与lncRNA-H19过表达组相比差异均有统计学意义(P均<0.05)。结论LncRNA-H19可通过特异性吸附miRNA-760调控nanog基因表达,从而促进NSCLC细胞增殖和迁移。Objective To explore the role of long non-coding RNA(lncRNA)-H19 in the proliferation and migration of non-small cell lung cancer(NSCLC)cells and the molecular mechanisms.Methods The expressions of lncRNA-H19 in 20 NSCLC tissues and paired non-tumor tissues,which were collected from Changhai Hospital of Naval Medical University(Second Military Medical University)from Oct.2015 to May 2016,were detected by real-time quantitative PCR(qPCR).We also examined lncRNA-H19 expressions in NSCLC cell lines A549 and NCI-H1299 and normal lung epithelial cell line BEAS-2B by qPCR.The proliferation and migration of A549 cells overexpressing lncRNA-H19 were detected by CCK-8 assay and Transwell assay,respectively.Bioinformatics analysis and duel-luciferase reporter assay were conducted to predict and confirm the interaction between microRNA(miRNA)-760 and lncRNA-H19.Western blotting analysis and RT-qPCR were performed to observe the influence of lncRNA-H19 overexpression on the expression of miRNA-760 and target gene nanog.MiRNA-760 was overexpressed in A549 cells,and its role in lncRNA-H19 promoting proliferation and migration of NSCLC cells was observed.Results The expressions of lncRNA-H19 in NSCLC tissues and A549 and NCI-H1299 cells were significantly upregulated compared with those in normal tissues and BEAS-2B cells(all P<0.01).Overexpression of lncRNA-H19 significantly improved the proliferation ability(P<0.05)and migration ability(P<0.01)of A549 cells compared with the negative control group.The results of starBase v3.0 showed that lncRNA-H19 could specifically adsorb miRNA-760,and duel-luciferase reporter assay showed that lncRNA-H19 directly bound to miRNA-760.Compared with the negative control group,overexpression of lncRNA-H19 significantly inhibited miRNA-760 expression in A549 cells(P<0.05)and promoted the expression of the downstream gene nanog at mRNA and protein levels(all P<0.01).Overexpression of miRNA-760 significantly inhibited lncRNA-H19-induced proliferation and migration of A549 cells(all P<0.05).Conclus

关 键 词：非小细胞肺癌 长链非编码RNA-H19 微RNA-760 NANOG基因 

分 类 号：R734.2[医药卫生—肿瘤]