摘要
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 gene silencing of Golgi α-mannosidase Ⅱ（GM Ⅱ） by RNA interference on the invasion and migration of human gastric carcinoma BGC-823 ceils. Methods： Four targeted locations were designed and four plasmid vectors expressing short hairpin RNA （shRNA） for gene silencing of GM Ⅱ were constructed, and the negative control plasmid vector was also constructed. The plasmids were transfected into human gastric carcinoma BGC-823 cells by LipofectAMINE 2000. The expression levels of GM Ⅱ mRNA and protein were detected by RT-PCR and Western blotting, respectively. The best plasmid for gene silencing of GM Ⅱ was selected. The invasive and migration capabilities of BGC-823 cells induced by gene silencing of GM Ⅱ were detected byTranswell invasion assay and scratch-wound assay, respectively. Results： Plasmid vector pGPU6/GFP/Neo-1303 was the best one for the gene silencing of GM H. The number of cells invading through the matrigel in the plasmid vector pGPU6/GFP/Neo-1303-transfected group was much lower than those in the blank control group and the pGPU6/GFP/Neo-shNC-transfected group （P 〈 0.05）. After the cells were cultured without serum for 24 h, the invasive and migration capabilities of the cells transfected with pGPU6/GFP/Neo-1303 were significantly weakened than those of the blank control group and the pGPU6/GFP/Neo-shNC-transfected group （P 〈 0.05）. Conclusion： The expressions of GM Ⅱ mRNA and protein in human gastric carcinoma BGC-823 cells can be silenced by RNA interference, which results in the inhibition of invasive and migration capabilities of BGC-823 cells. These results suggest thatGM Ⅱ might bea novel target for the treatment of human gastric carcinoma.
出处
《肿瘤》
CAS
CSCD
北大核心
2012年第6期402-407,共6页
Tumor
基金
国家自然科学基金资助项目(编号:30672431)
教育部高等学校博士学科点专项科研基金(编号:20060631006)
重庆市自然科学基金资助项目(编号:2010BB5361)
关键词
胃肿瘤
甘露糖苷酶类
基因沉默
肿瘤侵润
细胞迁移分析
Stomach neoplasms
Mannosidases
Gene silencing
Neoplasm invasiveness
Cell migration assays
