下调miRNA-21表达对人结肠癌HCT116细胞体外生长的影响  

Effects of down-regulation of microRNA-21 on the growth of human colon cancer cell line HCT116 in vitro

作　　者：陶弋婧 赵娟娟[1] 李永菊[1] 郭萌萌[1] 周涯[2] 秦娜琳[1] 郑静[1] 罗军敏[1] 徐林[1] 

机构地区：[1]遵义医学院免疫学教研室贵州省免疫学研究生创新基地 [2]遵义医学院医学物理学教研室,遵义563000

出　　处：《现代免疫学》2015年第1期6-12,共7页Current Immunology

基　　金：国家自然科学基金(81260398;31370918);教育部新世纪优秀人才支撑计划(NCET-12-0661)

摘　　要：研究下调miRNA-21表达对人结肠癌HCT116细胞体外生长的影响。利用反义核酸技术设计针对miRNA-21成熟体的ASO序列,构建pcDNA-6.2-miRNA-21-ASO真核表达载体(命名为p-miR-21-ASO);将重组载体p-miR-21-ASO体外瞬时转染HCT116细胞,real-time PCR检测细胞中miRNA-21的表达变化;CCK-8法及克隆形成实验检测HCT116细胞的增殖及克隆形成能力改变;划痕法观察HCT116细胞的体外迁移能力变化;western blot检测细胞中VEGF蛋白的表达变化。结果显示,p-miR-21-ASO载体能下调miRNA-21的表达(P<0.05);CCK-8及克隆形成实验结果显示HCT116细胞的增殖及克隆形成能力明显下降(P<0.05);划痕实验结果发现细胞的体外迁移能力削弱(P<0.05);western blot检测结果表明转染组细胞VEGF表达下调(P<0.05)。结果表明,下调miRNA-21能明显抑制HCT116细胞的体外生长,这可能与VEGF的表达降低有关。The aim of this study is to investigate the effects of downregulation of miR-21 on the growth of human colon cancer HCTll6 cells in vitro. Antisense oligo-deoxynucleotide technique was used to design miRNA-21 ASO sequence and construct a eukaryotic expression vector pcDNA-6.2-miRNA-2l-ASO（termed as p-miR-21-ASO）. The recombinant vector was transiently transfected into human colon cancer line HCTll6 cells. Then the relative expression level of miRNA-21 in HCT116 cells was determined using specific probe of real-time PCR assay. The proliferation of HCTll6 cells was detected by CCK-8 assay and clone formation assay. The migration of cells was analyzed by scratch assay. Finally, the expression of VEGF was measured by Western blot. The results showed that, compared with the controls, the expression level of miRNA-21 was significantly de creased in p-miR-21 ASO transfected group （P〈0.05）. The proliferation and clone formation capacity, as well as migration a bility, of cells in p-miR-21-ASO transfeeted group were significantly inhibited （P〈0.05）. Finally, the expression of VEGF in ceils was also significantly down-regulated （P〈0.05）. In conclusion, down-regulation of miRNA-21 could significantly inhibit the growth of human colon cancer HCTll6 cells in vitro, which might be closely related to the lower expression of VEGF.

关 键 词：MIRNA-21 结肠癌 真核表达 

分 类 号：R392.11[医药卫生—免疫学]