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目的探讨高尔基体囊泡转运蛋白(Golgi-vesicular transport protein)p115基因沉默对人胃癌BGC-823细胞侵袭能力的影响及其机制。方法采用脂质体法将质粒pGPU6/GFP/Neo/p115(p115 shRNA)和阴性对照质粒(shNC)分别转染至BGC-823细胞中,并设空白对照组。转染后48 h,采用RT-PCR、Westernblot和细胞免疫荧光法检测各组细胞中p115基因及蛋白表达的变化,及其对巨噬细胞游走抑制因子(Macrophage migration inhibitory factor,MIF)、基质金属蛋白酶-2(Matrix metallopro-teinase-2,MMP-2)、MMP-9基因和蛋白表达的调节;细胞划痕及Transwell小室试验检测细胞迁移及侵袭能力的变化。结果p115 shRNA组细胞中p115、MIF、MMP-2、MMP-9在基因和蛋白水平的表达均较shNC组和空白对照组明显下降(P<0.01);p115shRNA组细胞的划痕愈合能力明显低于两个对照组(P<0.01);p115 shRNA组的穿膜细胞数明显低于两个对照组(P<0.01)。结论在BGC-823细胞中,抑制P115的表达后,可能通过下调MIF、MMP-2、MMP-9的表达,抑制肿瘤细胞的侵袭运动。
Objective To investigate the effect of Golgi-vesicular transport protein p115 silencing on invasion of human gastric cancer BGC-823 cells and its mechanism. Methods Plasmid pGPU6 / GFP / Neo / p115 (p115 shRNA) and negative control plasmid (shNC) were transfected into BGC-823 cells by lipofectamine respectively. Forty-eight hours after transfection, the changes of p115 gene and protein expression in each group were detected by RT-PCR, Western blot and immunofluorescence, and their effects on macrophage migration inhibitory factor (MIF), matrix Matrix metallopro-teinase-2 (MMP-2), MMP-9 gene and protein expression regulation; cell scratches and Transwell chamber assay to detect changes in cell migration and invasion. Results The expression of p115, MIF, MMP-2 and MMP-9 in p115 shRNA group was significantly lower than that in shNC group and blank control group (P <0.01). The scratch healing ability of p115 shRNA group was significantly lower (P <0.01). The number of transmembrane cells in p115 shRNA group was significantly lower than that in the two control groups (P <0.01). Conclusion Inhibition of P115 expression in BGC-823 cells may inhibit the invasiveness of tumor cells by down-regulating the expression of MIF, MMP-2 and MMP-9.