RNA干扰ART1表达对小鼠结肠癌细胞增殖的影响及其机制探讨

Effect of ART1 gene silencing by RNA interference on the proliferation of mouse colon carcinoma cells and its possible mechanism

目的:通过短发夹RNA(short hairpin RNA,shRNA)干扰精氨酸特异性腺苷二磷酸核糖基化转移酶1(arginine-specific adenosinediphosphate-ribosyltransferase 1,ART1)基因的表达,研究沉默ART1基因对小鼠结肠癌CT26细胞增殖的影响及可能的机制。方法:细胞免疫荧光法检测ART1水平,以证实CT26细胞中有ART1的表达。用ART1-shRNA和阴性对照shRNA(negative control-shRNA,NC-shRNA)慢病毒感染CT26细胞后,分别采用RT-PCR和蛋白质印迹法检测各组细胞中ART1mRNA和ART1、RhoA、c-myc及c-fos蛋白的表达;CCK-8(cell countingkit-8)法分析各组细胞增殖情况。结果:CT26细胞中有ART1的表达。ART1-shRNA和NC-shRNA慢病毒成功感染细胞,获得稳定低表达ART1基因的CT26细胞株。与感染NC-shRNA慢病毒和未感染的CT26细胞相比,感染ART1-shRNA慢病毒的CT26细胞中ART1mRNA表达水平及ART1、RhoA、c-myc和c-fos的蛋白表达水平均显著降低(P<0.01),而细胞增殖抑制率明显升高(P<0.01)。结论:干扰ART1表达可抑制CT26细胞增殖,提示ART1在肿瘤生长、增殖过程中发挥重要作用,其机制可能与ART1基因沉默后RhoA及其下游因子c-myc和c-fos的表达被抑制有关。

Objective： To investigate the effect of ART1 （arginine-specific adenosinediphosphate-ribosyltransferase 1） gene silencing by shRNA （short hairpin RNA） interference on the proliferation ability of mouse colon cancer CT26 cells, and to explore its possible mechanism. Methods： It was confirmed that ART1 expression existed in CT26 cells by immunofluorescence assay. Lentivirus of ART1-shRNA was infected into mouse colon carcinoma CT26 cells. The CT26 cells uninfected or infected with a negative NC-shRNA （control-shRNA） served as the controls. The expression of ART1 mRNA was detected by RT-PCR, and the expressions of ART1, RhoA, c-myc, and c-fos proteins were examined by Western blotting. The cell proliferation in each group was measured by CCK8 （cell counting kit-8） assay. Results： It was determined that ART1 expression existed in the CT26 cells. Lentivirus of ART1-shRNA or NC-shRNA was infected into CT26 cells successfully, and the CT26 cell line with stable low-expression of ART1 was successfully established. Compared with CT26 cells infected with NC-shRNA lentivirus or those were un-infected, the expression of ART1 mRNA was significantly reduced in CT26 cells infected with ART1-shRNA lentivirus （P 0.01）, and the protein expression levels of ART1, RhoA, c-myc, and c-fos were all obviously decreased （P 0.01）. The inhibition rate of cell proliferation of CT26 cells infected with ART1-shRNA lentivirus was markedly increased compared with the control groups （P 0.01）. Conclusion： RNA interference targeting ART1 gene can inhibit the proliferation ability of mouse colon carcinoma CT26 cells. This effect probably associates with the down-regulation of the expressions of RhoA and its downstream effectors c-myc and c-fos after silencing the expression of ART1 gene.