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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。
To investigate the effect of miRNA-21 downregulation on the growth of human colon cancer HCT116 cells in vitro. The antisense oligonucleotide technology was used to design the ASO sequence of mature miRNA-21. The eukaryotic expression vector pcDNA-6.2-miRNA-21-ASO was named as p-miR-21-ASO. -ASO was transiently transfected into HCT116 cells in vitro and the expression of miRNA-21 was detected by real-time PCR. The proliferation and clonogenic capacity of HCT116 cells were detected by CCK-8 assay and colony formation assay. Scratch assay was used to detect the expression of HCT116 cells in vitro Migration ability changes; Western blot detection of VEGF protein expression changes. The results showed that the expression of miRNA-21 was down-regulated by p-miR-21-ASO vector (P <0.05). The results of CCK-8 and clonogenic assay showed that the proliferation and clonogenic capacity of HCT116 cells were significantly decreased (P <0.05) The experimental results showed that the migration ability of cells was weakened in vitro (P <0.05). Western blot results showed that the expression of VEGF in transfected cells was down-regulated (P <0.05). The results showed that downregulation of miRNA-21 can significantly inhibit the growth of HCT116 cells in vitro, which may be related to the decreased expression of VEGF.