沉默CDC6基因对结肠癌细胞增殖的影响及机制的初步研究

目的:探讨沉默CDC6表达对结肠癌细胞增殖的影响及可能作用的机制。方法:筛选高表达CDC6结肠癌细胞株，构建CDC6-shRNA慢病毒载体，感染结肠癌细胞，RT-qPCR、Western blotting验证。CCK8法、细胞克隆形成、流式细胞周期实验分别检测转染前后结肠癌细胞增殖及细胞周期变化。Western blotting检测各组ERK、pERK、AKT、pAKT蛋白水平。结果:CDC6在结肠癌SW620细胞中高表达，shRNA-CDC6慢病毒感染后，CDC6 mRNA及蛋白表达明显降低，敲减率达到82.2%（P＜0.01）。CDC6表达沉默后，CCK8实验结果显示，结肠癌细胞增殖速率明显下降，72小时抑制率达21.0%（P＜0.05）；细胞克隆实验也显示，干扰组结肠癌细胞克隆形成能力显著下降，相对对照组，干扰组细胞克隆数减少52.1%（P＜0.01）。流式细胞周期实验显示，相对对照组，干扰组细胞G0/G1期的细胞比例升高，从44.12%增加到55.03%（P＜0.05）。Western blotting实验结果显示，CDC6表达下调后，干扰组结肠癌细胞pERK、AKT及 pAKT蛋白表达显著减少（P＜0.05）。结论:沉默CDC6基因表达能抑制结肠癌细胞的增殖能力，可能与抑制ERK及AKT信号通路活性有关。

Effect and mechanism of silencing CDC6 gene on proliferation of colorectal carcinoma cells

Objective:To investigate the effect of silencing CDC6 expression on the biological behavior of colorectal carcinoma cells and its possible mechanism.Methods:Colorectal carcinoma cells with high expression of CDC6 were screened,CDC6 was knocked down in colorectal carcinoma cells by constructing the shRNA interference carrier GV115．The lentivirus was prepared and infected with SW620 cells，The transfected cells were verified by RT-qPCR and Western blotting.CCK8 method,cell clone formation and flow cytometry were used to detect the proliferation and cell cycle changes of colon cancer cells before and after transfection.The protein levels of ERK,pERK,AKT and pAKT were detected by Western blotting.Results:CDC6 was highly expressed in colorectal carcinoma SW620 cells.After infection with shRNA-CDC6 lentivirus,the expression of CDC6 mRNA and protein was significantly decreased,and the knockdown rate was 82.2%(P＜0.01).After silencing CDC6 expression,CCK8 assay showed that the proliferation rate of colon cancer cells decreased significantly,and the inhibition rate was 21.0% at 72 h(P＜0.05).Cell cloning assay also showed that the colony forming ability of colon cancer cells in the interference group decreased significantly,compared with the control group,the number of colon cancer cells in the interference group decreased by 52.1%(P＜0.01).Flow cytometry showed that compared with the control group,the proportion of G0/G1 phase cells in the interference group increased from 44.12% to 55.03%(P＜0.05).Western blotting analysis showed that the expression of pERK,AKT and pAKT protein in colon cancer cells in the interference group decreased significantly after the down-regulation of CDC6 expression(P＜0.05).Conclusion:Silencing CDC6 gene expression can inhibit the proliferation of colon cancer cells,which may be related to the inhibition of ERK and AKT signaling pathway.