miRNA-4262通过靶向神经调节蛋白1调控非小细胞肺癌细胞增殖、侵袭、迁移的作用机制  

Mechanism of miRNA-4262 regulating the proliferation,invasion and migration of non-small cell lung cancer cells by targeting NRG1

作　　者：王旭[1] 徐文举 袁五营[1] WANG Xu;XU Wenju;YUAN Wuying(Department of Minimally Invasive Surgery,He’nan Provincial Chest Hospital,Zhengzhou 450000,He’nan,China)

机构地区：[1]河南省胸科医院微创外科

出　　处：《癌症进展》2020年第2期133-137,共5页Oncology Progress

基　　金：2017年度河南省科技攻关项目(172102310305)

摘　　要：目的探讨miRNA-4262通过靶向神经调节蛋白1(NRG1)调控非小细胞肺癌(NSCLC)细胞增殖、侵袭、迁移的作用机制。方法采用实时定量聚合酶链反应(qRT-PCR)、蛋白质印迹法(Western blot)检测正常肺上皮细胞BEAS-2B及NSCLC细胞A549、H1299、H1650中miRNA-4262、NRG1的表达水平,Western blot检测转染后A549细胞中细胞周期蛋白D1(cyclin D1)、p21、p27、基质金属蛋白酶(MMP)2、MMP9、MMP14的表达水平,噻唑蓝(MTT)法检测细胞的增殖情况,Transwell实验检测细胞的侵袭和迁移能力,双荧光素酶报告实验及Western blot分析miRNA-4262与NRG1的关系。结果与正常肺上皮细胞BEAS-2B相比,NSCLC细胞株A549、H1299、H1650中miRNA-4262的表达水平明显升高,NRG1 mRNA及NRG1蛋白表达水平明显降低(P﹤0.01),选择A549细胞进行后续实验。与anti-miRNA-NC组比较,抑制miRNA-4262表达后,anti-miRNA-4262组48、72 h的细胞活性、侵袭和迁移细胞数目及cyclin D1、MMP2、MMP9、MMP14蛋白表达水平均明显降低,p21、p27蛋白表达水平均明显升高(P﹤0.01)。与pcDNA组比较,过表达NRG1后,pcDNA-NRG1组中NRG1、p21蛋白表达水平均明显升高,48、72 h细胞活性、侵袭和迁移细胞数目及cyclin D1、MMP2、MMP9蛋白表达水平均明显降低(P﹤0.01)。TargetScan生物信息学软件预测结果显示,miRNA-4262与NRG1的3'UTR存在特异性结合位点,双荧光素酶报告实验和Western blot进一步证实miRNA-4262与NRG1直接结合负向调控NRG1的表达。与anti-miRNA-4262+si-NC组比较,在稳定低表达miRNA-4262的细胞中沉默NRG1基因后,anti-miRNA-4262+si-NRG1组中NRG1、p21蛋白表达水平均降低,48、72 h的细胞活性、侵袭和迁移细胞数目及cyclin D1、MMP2、MMP9蛋白表达水平均升高(P﹤0.05)。结论miRNA-4262通过靶向抑制NRG1的表达,促进NSCLC的增殖、侵袭和迁移。Objective To explore the mechanism of miRNA-4262 regulating the proliferation,invasion and migration of non-small cell lung cancer(NSCLC)cells by targeting neuroregulatory protein 1(NRG1).Method Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blot were used to detect the expression of miRNA-4262 and NRG1 in normal lung epithelial cells BEAS-2B and in NSCLC cells A549,H1299 and H1650.Western blot was used to detect the expression of cyclin D1,p21,p27,matrix metalloproteinase(MMP)2,MMP9 and MMP14 in transfected A549 cells.Methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay was utilized to detect cell proliferation,transwell assay was employed to determine cell invasion and migration,dual-luciferase reporter assay and Western blot were used to investigate the relationship between miRNA-4262 and NRG1.Result Compared with normal lung epithelial cells BEAS-2B,the expression levels of miRNA-4262 in NSCLC cell lines A549,H1299 and H1650 increased significantly,and the level of NRG1 mRNA and proteins decreased significantly(P<0.01).A549 cells were selected for subsequent experiments.Compared with anti-miRNA-NC group,after inhibiting the expression of miRNA-4262,the cell viability at 48 h and 72 h,the number of invasive and migrating cells,as well as expression of cyclin D1,MMP2,MMP9 and MMP14 in anti-miRNA-4262 group decreased significantly,while the levels of p21 and p27 increased significantly(P<0.01).Compared with pcDNA group,after overexpression of NRG1,the levels of NRG1 and p21 proteins in pcDNA-NRG1 group increased significantly,while the cell viability at 48,72 h,the number of invasive and migrating cells,the expression of cyclin D1,MMP2 and MMP9 decreased significantly(P<0.01).TargetScan bioinformatics software predicted that there was a specific binding site between miRNA-4262 and NRG13'UTR,dual-luciferase reporter assay and Western blot further confirmed that miRNA-4262 and NRG1 directly combined to negatively regulate the expression of NRG1.NRG1 gene was silenced in cells with stable and

关 键 词：miRNA-4262 NRG1 非小细胞肺癌 增殖 侵袭 转移 

分 类 号：R734.2[医药卫生—肿瘤]