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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。
Objective To investigate the effect of c-MetshRNA expression vector on the proliferation and invasion of human melanoma A375 cells. Methods pGenesil-1 plasmid was used as vector and c-Met as target gene. The shRNA expression vector was constructed and transfected into human melanoma A375 cells cultured in vitro. The morphological changes of tumor cells after interference were observed under inverted phase contrast microscope; the expression levels of c-Met gene and protein in transfected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot; The motility of the transfected cells was detected by MTT assay with azozolium salt, and the invasive ability of transfected cells was measured by Transwell chamber method. Results The pGenesil-1-c-MetshRNA recombinant plasmid was constructed and successfully transfected into A375 cells. After transfection, A375 cells shrink and round. RT-PCR showed that the expression of c-Met mRNA was significantly decreased after transfection, which was 48 h, which was a decrease of nearly 64%. Western blot confirmed that the expression of the protein after transfection decreased by nearly 65%; MTT assay showed that the cell viability was reduced to (68.25). ±4.4%; Transwell chamber assay transfected cells in vitro significantly decreased invasive ability. Conclusion pGenesil-1-c-MetshRNA recombinant plasmid significantly down-regulates the expression of c-Met in melanoma cells and inhibits the proliferation and invasion of tumor cells.