hsa-miR-448过表达下调HIF-1α对结肠癌细胞SW480作用及机制研究  

Effect and mechanism of overexpression of hsa-miR-448 by down-regulating HIF-1α on colon cancer cell line SW480

作　　者：歧红阳[1] 王云溪[1] 肖占宇[1,2] 王志民[1] 吴凤丽 董志超[1] 王蕾 QI Hong-Yang;WANG Yun-Xi;XIAO Zhan-Yu;WANG Zhi-Min;WU Feng-Li;DONG Zhi-Chao;WANG Lei(Department of Gastroenterology,Xinxiang Central Hospital,Xinxiang 453000,China)

机构地区：[1]新乡市中心医院消化内科,新乡453000 [2]新乡市第一人民医院体检科,新乡453000 [3]郑州大学第一附属医院,郑州450052

出　　处：《中国免疫学杂志》2019年第19期2350-2356,共7页Chinese Journal of Immunology

基　　金：河南省自然科学基金面上项目(No.162300410220)

摘　　要：目的:本研究旨在探讨小RNA-448(miR-448)在结肠癌发生和发展中的作用及其潜在的分子机制。方法:先用基因预测软件:miRanda、TarBase和TargetScan预测miR-448的靶基因,并用荧光素实验验证靶向关系;然后miR-448 mimic与pcDNA-HIF-1α单独或联合转染SW480细胞,并用RT-PCR检测miR-448和缺氧诱导因子1α(HIF-1α)的表达,Transwell检测细胞侵袭情况,划痕实验检测细胞的迁移能力,蛋白印迹检测HIF-1α、E-cadherin、Vimentin、N-cadherin;P38和Hsp27的蛋白表达;miR-448 inhibitor转染结肠癌细胞SW480、10μmol/L SB203580处理结肠癌细胞SW480单独或联合进行,RT-PCR检测miR-448的表达,蛋白印迹检测HIF-1α、P38和Hsp27的表达,Transwell检测细胞侵袭;裸鼠皮下注射miR-448 mimic转染结肠癌细胞SW480,检测30 d时移植瘤重量,RT-PCR检测miR-448和HIF-1α的表达,免疫组化检测Vimentin的表达,蛋白印迹法检测P38和Hsp27的表达。结果:miR-448与HIF-1α在3′UTR区存在结合位点,miR-448和HIF-1α存在靶向关系,并且miR-448对HIF-1α表达具有抑制作用;SW480细胞过表达miR-448后,HIF-1α表达量下调,细胞侵袭和迁移速度减慢,E-cadherin的表达显著上调,而N-cadherin和Vimentin的表达显著下调;低表达miR-448后,HIF-1α、P38和Hsp27表达量显著增加,细胞侵袭数目显著增多;小鼠转染MIR-448 mimic后,移植瘤重量显著减轻,miR-448表达量显著上调,HIF-1α表达量显著下调,Vimentin阳性比率明显降低,P38和Hsp27表达量显著下调。结论:在结肠癌细胞SW480中,过表达hsa-miR-448下调HIF-1α来减弱细胞的侵袭迁移能力以及抑制上皮间质转化,从而抑制结肠癌的发生和发展。Objective:To investigate the effect and mechanism of overexpression of hsa-miR-448 by down-regulating HIF-1αon colon cancer cell line SW480.Methods:The target genes of miR-448 were predicted by gene prediction software:miRanda,TarBase and TargetScan,and the targeting relationship was verified by fluorescein experiments;then,miR-448 mimic and pcDNA-HIF-1αwere transfected into SW480 cells alone or in combination,and the expression of miR-448 and HIF-1αwas detected by RT-PCR,the cell invasion was detected by Transwell,and the migration ability of cells was detected by scratch test.HIF-1α,E-cadherin,Vimentin,N-cadherin were detected by immunoblot.Subsequently,miR-448 inhibitor was transfected into colon cancer cells SW480,10μmol/L SB203580 treated colon cancer cells SW480.The expression of miR-448 was detected by RT-PCR.The expression of HIF-1α,P38 and Hsp27 was detected by Western blot,and cell invasion was detected by Transwell;finally,nude mice were subcutaneously injected with miR-448 mimic to transfect colon cancer cells SW480,and the tumor weight was measured by weighting method for 30 days.The expression of miR-448 and HIF-1αwas detected by RT-PCR.The expression of Vimentin was detected by immunohistochemistry.The expression of P38 and Hsp27 was detected by Western blot.Results:miR-448 and HIF-1αhad binding sites in the 3′UTR region,miR-448 and HIF-1αhad a targeting relationship,and miR-448 had an inhibitory effect on HIF-1αexpression;after overexpression of miR-448 in SW480 cells,the expression of HIF-1αwas down-regulated;the number of cell invasion and migration was significantly decreased;the expression of E-cadherin was significantly up-regulated,while the expression of N-cadherin and Vimentin was significantly down-regulated.After low expression of miR-448,the expression levels of HIF-1α,P38 and Hsp27 were significantly increased,and the number of cell invasion was significantly increased.After transfected with miR-448 mimic,the tumor weight was significantly reduced,the expression of miR-44

关 键 词：miR-448 HIF-1Α 结肠癌 侵袭 迁移 EMT 

分 类 号：R734.2[医药卫生—肿瘤]