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目的:探讨RNA干扰沉默高尔基体α-甘露糖苷酶Ⅱ(Golgiα-mannosidaseⅡ,GMⅡ)基因表达对人胃癌BGC-823细胞侵袭和迁移的影响。方法:为沉默GMⅡ基因,构建了4个分别针对不同靶位点的短发夹RNA(shorthairpinRNA,shRNA)质粒表达载体,同时合成阴性对照质粒表达载体。应用LipofectAMINE2000将质粒转染入BGC-823细胞。分别采用RT-PCR和蛋白质印迹法检测转染后GMⅡmRNA和蛋白表达的变化,选出沉默效果最佳的质粒。应用Transwell侵袭实验和划痕损伤实验分别检测GMⅡ基因沉默对人胃癌细胞BGC-823侵袭和迁移能力的影响。结果:质粒载体pGPU6/GFP/Neo-1303沉默GMⅡ基因的效果最好。转染pGPU6/GFP/Neo-1303组穿透小室基质的细胞数明显少于空白对照组和转染阴性对照组(P<0.05)。无血清培养液培养24h后,转染pGPU6/GFP/Neo-1303组细胞的迁移能力较空白对照组和转染阴性对照组明显降低(P<0.05)。结论:RNA干扰技术可沉默人胃癌BGC-823细胞中GMⅡmRNA和蛋白的表达,从而抑制BGC-823细胞的侵袭和迁移能力。GMⅡ可能成为胃癌治疗的新靶点之一。
OBJECTIVE: To investigate the effect of silencing Golgiα-mannosidase Ⅱ (GMⅡ) gene expression on the invasion and migration of human gastric cancer cell line BGC-823 by RNA interference. Methods: To silence the GMⅡ gene, four shorthairpinRNA (shRNA) plasmid expression vectors targeting different target sites were constructed and a negative control plasmid vector was constructed. Plasmids were transfected into BGC-823 cells using LipofectAMINE2000. RT-PCR and Western blotting were used to detect the expression of GMⅡmRNA and protein after transfection, and the best plasmid for silencing was selected. Transwell invasion assay and scratch injury assay were used to detect the effect of GMⅡ gene silencing on the invasion and migration of human gastric cancer cell line BGC-823. Results: The plasmid pGPU6 / GFP / Neo-1303 silenced the GMⅡ gene. The number of transfected cells in pGPU6 / GFP / Neo-1303 group was significantly less than that in blank control group and transfected negative control group (P <0.05). After cultured in serum-free medium for 24 h, the migration ability of pGPU6 / GFP / Neo-1303 group was significantly lower than that of blank control group and transfected negative control group (P <0.05). Conclusion: RNAi can silence the expression of GMⅡmRNA and protein in human gastric cancer cell line BGC-823 and inhibit the invasion and migration of BGC-823 cells. GM Ⅱ may become a new target of gastric cancer treatment.