RNAi沉默c-Met基因对黑素瘤A375细胞生物学行为的影响

目的探讨c-MetshRNA表达载体对人黑素瘤A375细胞增殖、侵袭能力的影响。方法以pGenesil-1质粒为载体,以c-Met为靶基因,构建shRNA表达载体,并将其转染至体外培养的人黑素瘤A375细胞中。倒置相差显微镜下观察经干扰后瘤细胞的形态学变化;采用反转录聚合酶链反应(RT-PCR)和Western blot法分别检测转染细胞c-Met基因和蛋白的表达水平;四甲基偶氮唑盐微量酶反应比色法(MTT法)检测转染后细胞的生长活力;Transwell小室法测定转染细胞体外侵袭能力。结果构建了pGenesil-1-c-MetshRNA重组质粒,并成功转染A375细胞。转染后A375细胞缩小,变圆。RT-PCR显示转染后c-Met mRNA的表达显著降低,以48h为著,下降近64%;Western blot证实转染后蛋白的表达下降近65%;MTT法显示细胞的活力降低为(68.25±4.4)%;Transwell小室测定转染细胞体外侵袭能力明显下降。结论 pGenesil-1-c-MetshRNA重组质粒明显下调c-Met在黑素瘤细胞中的表达,并抑制肿瘤细胞增殖及其侵袭能力。

The Effects of RNA Interference to c-Met Gene on Cytobiology Behavior of Human Melanoma Cell Line A375

Objective To investigate the effects of RNA interference to c-Met gene on proliferation and invasion of human melanoma cell line A375. Methods The pGenesil-1 plasmid was used to constructed shRNA of c-Met and was transfected into A375 cells. Morphological changes of transfected cells were observed under inversed microscopy. The mRNA and protein level of c-Met were detected by RT-PCR and Western blot. The proliferating and invading ability of transfected A375 cells were evaluated by MTT and Transwell cabin separately. Results Transfected cells with pGenesil-1-c-Met shRNA showed round and small. Results of RT-PCR and western blot revealed c-Met mRNA was remarkably decreased,especially 48h later was decreasing nearly 64% while level of c-Met protein decreased nearly 65% after transfection. MTT confirmed c-Met shRNA could suppress the growth of A375 cells to(68.25±4.4)%. The invasion of A375 cells were significantly decreased. Conclusion pGenesil-1-c-Met shRNA could significantly inhibit c-Met expression,suppress the growth of A375 cells and decrease their invasion.