慢病毒介导miR-222稳定沉默结肠癌细胞系的构建及其生物学作用

Construction of colon cancer cell line with lentivirus-mediated MiR-222 stable silence and its biological effect

目的构建稳定沉默miR-222结肠癌HCT-116细胞系,探讨miR-222对结肠癌细胞增殖、周期、凋亡及迁移能力的影响。方法选取HCT-116细胞,应用TuD RNA(Tough Decoy RNA)构建TUD-hsa-miR-222-3p Inhibitor慢病毒载体,分别转染miR-222 Inhibitor(miR-222 I)和阴性对照(NC),未感染慢病毒细胞为正常组(HCT-116)。采用MTS法检测细胞活力及Annexin V-FITC检测细胞凋亡,流式细胞仪检测细胞周期;Transwell小室模型检测细胞迁移能力。结果miR-222 Inhibitor转染HCT-116细胞后,经荧光显微镜、RT-PCR证实转染成功。与NC组和正常组相比,miR-222 I组细胞活力明显下降;处于S期的细胞比例明显降低,早期细胞凋亡率显著升高。细胞迁移能力明显受到抑制。结论miR-222基因的沉默能降低结肠癌细胞活力和阻滞周期进程、促进凋亡及抑制细胞迁移能力。

Objective To construct a colon cancer HCT-116 cell line with stable silencing of miR-222,and to explore the effect of miR-222 on colon cancer cell proliferation,cycle,apoptosis and migration ability.Method HCT-116 cells were selected and TuD RNA(Tough Decoy RNA)was used to construct TUD-hsa-miR-222-3p Inhibitor lentiviral vector,which was transfected into miR-222 Inhibitor(miR-222 I)and negative control(NC).The normal group was not infected with lentivirus cells(HCT-116).MTS method was used to detect cell viability and Annexin V-FITC was used to detect cell apoptosis;flow cytometry was used to detect cell cycle;tran-swell cell model was used to detect cell migration ability.Results After miR-222 inhibitor was transfected into HCT-116 cells and the transfection was confirmed by fluorescence microscopy and RT-PCR.Compared with that of NC group and normal group,cell viability of miR-222 I group was significantly reduced;the proportion of cells in the S phase was significantly reduced,and the early apoptosis rate was significantly increased.The cell migration ability was obviously inhibited.Conclusion Silencing of miR-222 gene can reduce colon cancer cell viability and block cycle progression,promote apoptosis and inhibit cell migration.