靶向转录信号传导子与激活子-3基因的RNAi对人结肠癌细胞HCT116侵袭能力的影响

The Effect of RNAi Targeting STAT-3 Gene on the Invasion of Human Colonic Carcinoma Cell Line HCT116

目的探讨抑制靶向转录信号传导子与激活子-3(STAT-3)基因表达对结肠癌细胞增殖以及侵袭转移行为的影响。方法实验分三组:空白对照组、转染NC-siRNA(阴性对照组)及转染特异性STAT-3-siRNA组(RNAi组)。人结肠癌细胞HCT116瞬时转染特异性siRNA,24、48、72 h后MTT法检测三组细胞的增殖活性,48 h后RQPCR检测STAT-3基因mRNA表达。RQ-PCR及细胞免疫化学法检测MMP-9在mRNA和蛋白水平的表达,Transwell小室法检测细胞体外侵袭能力。结果 RNAi组与其他两组相比细胞增殖受抑明显(均P<0.01);细胞生长减慢,RNAi组较阴性对照组STAT-3mRNA下降了71.91%(P<0.01)。RNAi组与其他两组相比MMP-9在mRNA和蛋白水平表达均明显下降(均P<0.01);侵袭实验RNAi组较空白对照组侵袭力下降了49.75%(P<0.01),而两对照组之间差异无统计学意义(P>0.05)。结论下调STAT-3基因表达可有效抑制结肠癌细胞的增殖和侵袭转移,抑制MMP-9在mRNA和蛋白水平的表达。沉默STAT-3基因抑制结肠癌细胞侵袭能力的机制之一可能是通过下调MMP-9途径。

Objective To approach the effect of RNAi targeting STAT-3 on the proliferation and invasion of colonic carcinoma cells.Methods Three groups were divided into the RNAi group,the negative control group and the blank group.For 72 hours after transfecting HCT116 cells with specific STAT-3 siRNA or N.C.FAM siRNA,MTT method was used to detect the proliferation of the cells every day three days afer transfection and the cell growth curve was made.The level of STAT-3 mRNA in cells was measured by RQ-PCR. Forty eight hours after transfecting,the level of MMP-9 mRNA in cells was measured by RQ-PCR, immuocytochemical method was used to detect the expression of MMP-9 protein,the invasiveness of HCT116 cells in vitro was measured quantitatively by the invasion test of transwell chamber.Results Compared with the two control groups,cell proliferation in the siRNA group was obviously suppressed,the cells in the siRNA group grew slowly（P〈0.01）.The expression of STAT-3 mRNA in cells in the siRNA group reduced by 71.91%（P〈0.01）,but in the negative control group and the blank group there was no obvious difference（P〉0.05）.The expression of MMP-9 mRNA and protein in the siRNA group was obvi- ously suppressed compared with that in the blank group and the negative control group（P〈0.01）.The invasiveness of HCT116 cells also reduced by 49.75%（P〈0.01）,but in the negative control group and the blank group there was no obvious difference（P〉0.05）.Conclusions Silencing STAT-3 can effectively inhibit the proliferation,invasiveness and the expression of MMP-9 in mRNA and the protein level of human colonic carcinoma.Silencing STAT-3 may become a new way of therapy for human colonic carcinoma. Down regulation of MMP-9 may be one of the important mechanisms to inhibit the invasiveness of colonic carcinoma.