ART1基因沉默对小鼠结肠癌CT26细胞转移潜能的影响及作用机制的研究

Effect of ART1 gene silencing on metastatic potential of mouse colon cancer CT26 cells and its mechanism

目的:通过沉默精氨酸特异性单-ADP-核糖基化作用转移酶-1(arginine-specic mono-ADP-ribosytransferase-1,ART1)基因的表达,探讨其对小鼠结肠癌CT26细胞转移潜能的影响及可能的作用机制。方法:采用慢病毒转染系统将特异性针对ART1基因的ART1-shRNA转入结肠癌CT26细胞中,在荧光显微镜下观察转染效率;分别采用RT-PCR和蛋白质印迹法鉴定ART1基因沉默的效果;采用Transwell小室侵袭实验和黏附实验观察ART1基因沉默对CT26细胞侵袭及黏附能力的影响;明胶酶谱法测定ART1基因沉默对CT26细胞中基质金属蛋白酶-2(matrix metall oproteinase-2,MMP-2)及MMP-9蛋白活性的影响;蛋白质印迹法检测对ART1、RhoA(Ras homolog gene family,member A)、ROCK1(Rho-associated coiled-coil contanining kinase 1)、MMP-2和MMP-9蛋白表达的影响。结果:成功获得了ART1基因沉默的稳定细胞株;ART1-shRNA转染组CT26细胞的侵袭及黏附抑制率分别为39.63%和40.70%,与空白对照组相比,ART1基因沉默后CT26细胞侵袭和黏附能力降低(P<0.05);MMP-2和MMP-9蛋白的活性降低,RhoA、ROCK1、MMP-2及MMP-9蛋白的表达量均被下调(P<0.05)。结论:ART1基因沉默可降低CT26细胞的侵袭和黏附能力,可能与ART1基因沉默后下调RhoA、ROCK1、MMP-2和MMP-9的表达以及MMP-2和MMP-9蛋白的活性有关,提示ART1基因在结肠癌侵袭和转移中发挥重要的作用。

Objective： To investigate the effect of ART1 （arginine-specific mono-ADP-ribosyltransferase-1） gene silencing on metastatic potential of mouse colon cancer CT26 cells, and to explore its possible mechanism. Methods： Lentivirus of ART1 -shRNA was infected into CT26 cells and the infection efficiency was observed by fluorescence microscope. The expression levels of ART1 mRNA and protein were detected by RT-PCR and Western blotting, respectively. The invasion and adhesion potencies were observed by Transwell chamber and cell matrix adhesion assay, respectively. The activity levels of MMP-9 （matrix metalloproteinase-9） and MMP-2 were determined by gelatin zymography assay. The expression levels of ART1, RhoA （Ras homolog gene family, member A）, ROCK1 （Rho-associated coiled-coil contanining kinase 1）, MMP-9 and MMP-2 were detected by Western blotting. Results： The stable cell lines infected with lentivirus of ARTI-shRNA were obtained successfully. After transfection of ART1 -shRNA, the expression levels of ART1 mRNA and protein were decreased in CT26 cells. As compered with the blank control group, the inhibitory rates of invasion and cell matrix adhesion of the cells in ART1 -shRNA group （39.63% and 40.70%） were sinificantly diseased （both P 〈 0.05）. The expression levels of RhoA, ROCK1, MMP-2 and MMP-9 proteins and the activities of MMP-2 and MMP-9 proteins in ARTI-shRNA group were decreased （P 〈 0.05）. Condusion： ART1 gene silencing in CT26 cells can inhibite the abilities of cell invasion and cell matrix adhesion, which may be associated with ART1 gene silencing and the blocking-activities of RhoA, ROCK1, MMP-9 and MMP-2. All of these suggest that ART1 gene probably plays a significant role in invasion and metastasis of colon cancer.