腺病毒介导miR-99a过表达抑制人结肠癌HCT-8细胞的生长抑制及其作用机制

Effect of adenovirus mediated overexpression of miR-99a on growth of human colon cancer HCT-8 cells and its mechanism

目的探讨miR-99a过表达对人结肠癌HCT-8细胞的生长抑制及其作用机制。方法构建miR-99a的重组腺病毒Ad5-miR-99a,并感染人结肠癌HCT-8细胞。采用甲基化特异性PCR检测miR-99a、抑癌基因SOCS1和P16的甲基化水平;采用实时荧光定量PCR和Western Blot检测miR-99a、DNAC5胞嘧啶甲基转移酶、SOCS1和P16的表达情况;采用MTT法、细胞集落形成和流式细胞术检测HCT-8细胞的体外增殖凋亡情况;采用裸鼠体内荷瘤实验检测HCT-8细胞体内成瘤性。结果成功构建miR-99a腺病毒载体Ad5-miR-99a。Ad5-miR-99a感染HCT-8细胞后,Ad5-miR-99a组细胞中miR-99a表达显著高于空载腺病毒组和空白对照组(P<0.05),而其甲基化水平显著低于空载腺病毒组和空白对照组(P<0.05);Ad5-miR-99a组细胞中DNAC5胞嘧啶甲基转移酶表达显著低于空载腺病毒组和空白对照组(P<0.05);Ad5-miR-99a组细胞中SOCS1和P16表达显著高于空载腺病毒组和空白对照组(P<0.05),而其甲基化水平显著低于空载腺病毒组和空白对照组(P<0.05)。MTT法、细胞集落形成、流式细胞术和裸鼠体内荷瘤实验显示,Ad5-miR-99a组细胞的体外增殖和体内成瘤能力显著低于空载腺病毒组和空白对照组(P<0.05)。结论 miR-99a可显著抑制人结肠癌HCT-8细胞的体外增殖和体内成瘤能力,其机制可能与miR-99a过表达导致DNMT1表达升高,降低抑癌基因SOCS-1和P16甲基化程度,使得SOCS-1和P16高表达,从而发挥抑制肿瘤细胞生长的作用。

Objective To investigate the inhibitory effect of miR- 99 a overexpression on human colon cancer HCT- 8 cell line and its potential mechanism. Methods miR- 99 a recombinant adenovirus Ad5- miR- 99 a was constructed and to infect human colon cancer HCT-8 cells. The methylation levels of P16, SOCS1 and miR-99 a were detected by methylation specific PCR（MSP）. The expressions of miR- 99 a, DNMT1, SOCS1 and P16 were inspected by real- time PCR and Western Blot. Proliferation and apoptosis of HCT- 8 cells were detected by MTT assay, cell colony formation and flow cytometry. In vivo tumor forming ability of HCT- 8cells were determined by nude mice. Results miR-99 a adenovirus vector Ad5-miR-99 a was successfully constructed. After infection, the expression of miR-99 a in Ad5-miR-99 a group was significantly higher than those in normal and control Ad-blank group（P〈0.05）, and its methylation level was significantly lower than those of normal and control Ad-blank（P〈0.05）. The expression of DNMT1 in Ad5-miR-99 a group was significantly lower than those in normal and control Ad-blank group（P〈0.05）. The expressions of P16 and SOCS1 in Ad5- miR- 99 a group were significantly higher than those in normal and control Ad- blank group（P〈0.05）,and the methylation levels were significantly lower than those in normal and control group（P〈0.05）. MTT method, cell colony formation, flow cytometry and in vivo tumor forming ability experiments showed that the proliferation of Ad5- miR- 99 a cells in vitro and in vivo were significantly lower than those in Ad- blank and control normal group（P〈0.05）. Conclusion Mi R- 99 a can significantly inhibit the proliferation of human colon cancer HCT-8 cells in vitro and in vivo. The mechanism may be related to the overexpression of miR-99 a, which leads to the increase of DNMT1 expression and decrease the level of tumor suppressor gene SOCS-1 and P16, which can inhibit the growth of tumor cells by SOCS-1 and P16.