高尔基体囊泡转运蛋白P115基因沉默对胃癌细胞凋亡的影响及其机制

Effect of Golgi body vesicular transporter P115 gene silencing on apoptosis of gastric cancer cells and relevant mechanism

目的探讨高尔基体囊泡转运蛋白P115基因沉默对胃癌BGC-823细胞凋亡的影响及其可能的相关机制。方法将含p115基因的重组质粒p115 shRNA-1318转染BGC-823细胞,并设shNC(阴性对照)转染组和空白对照组,免疫荧光显微镜检测转染效果;RT-PCR法检测细胞中p115和巨噬细胞游走抑制因子(Macrophage migration inhibitory factor,MIF)基因mRNA的转录水平;Western blot检测细胞中P115、MIF及核内肿瘤抑制蛋白质P53的表达水平;MTT法检测细胞的增殖活力;流式细胞术检测细胞凋亡及细胞周期的分布。结果转染后48 h,荧光显微镜下可见绿色荧光蛋白的表达,表明转染成功;与对照组相比,p115 shRNA-1318组细胞中的p115和MIF基因mRNA的转录水平及蛋白的表达水平均明显降低(P<0.05),细胞核内P53蛋白的表达水平明显增加(P<0.05),细胞的增殖活力明显下降(P<0.05),细胞凋亡数量明显增加(P<0.05),G1/G2期细胞数量明显增多,S期细胞数量明显减少(P<0.05)。结论沉默p115基因可促进胃癌细胞的凋亡,其调控机制可能是通过调控MIF因子来完成的。

Objective To investigate the effect of Golgi body vesicular transporter P115 gene silencing on apoptosis of gastric cancer BGC-823 cells as well as the relevant mechanism.Methods BGC-823 cells were transfected with recombinant plasmid p115 shRNA-1318 containing p115 gene,setting negative control（shNC） and blank control groups at the same time,and determined for transfection efficacy by IFA,for transcriptions of p115 and macrophage migration inhibitory factor（MIF） mRNAs by RT-PCR,for expression levels of P115,MIF and intranuclear tumor-inhibiting protein P53 by Western blot,for proliferation activity by MTT method,and for apoptosis and distribution of cell cycles by flow cytometry.Results The expression of green fluorescent protein（GFP） was observed in BGC-823 cells 48 h after transfection,indicating a successful transfection.Compared with those in control groups,both the mRNA transcription levels and protein expression levels of p115 and MIF genes in the cells transfected with recombinant plasmid p115 shRNA-1318 decreased significantly（P 0.05）,while the expression level of intranuclear P53 protein increased significantly（P 0.05）;the proliferation activity of BGC-823 cells transfected with p115 shRNA-1318 decreased significantly（P 0.05）,while the apoptosis level increased significantly（P 0.05）;however,the number of cells transfected with p115 shRNA-1318 at G1 / G2 phases increased significantly,while that at S phage decreased significantly（P 0.05）.Conclusion The p115 gene silencing promoted the apoptosis of gastric cancer cells by a potential mechanism of regulating MIF.