摘要
目的:观察高尔基体磷蛋白3(Golgi phosphoprotein 3,GOLPH3)基因过表达后胃癌细胞增殖的情况.方法:构建GOLPH3基因过表达重组慢病毒颗粒pGC-FU-GOLPH3-EGFP,用慢病毒法感染胃癌细胞株SGC-7901,利用荧光显微镜(Axiovert200)观察感染后细胞内EGFP表达的情况,利用流式细胞技术检测慢病毒的感染效率,采用实时荧光定量PCR(quantitative real-time polymerase chain reaction)和蛋白质印迹(Western blot)技术分别在基因水平和蛋白水平检测GOLPH3在SGC-7901-GOLPH3组(感染pGC-FU-GOLPH3-EGFP质粒的SGC-7901)、SGC-7901-empty-vector组(感染pGC-FU-EGFP质粒的SGC-7901)和SGC-7901-blank组(未感染慢病毒的SGC-7901)的表达水平;以四唑蓝比色法(MTT)检测GOLPH3基因过表达后胃癌细胞增殖的情况;Western blot技术检测胃癌细胞SGC-7901稳转细胞株中哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的表达水平.结果:建立了慢病毒感染率接近100%的胃癌细胞株SGC-7901,在体外能够长期稳定表达绿色荧光蛋白;SGC-7901-GOLPH3组GOLPH3mRNA和蛋白表达水平明显高于SGC-7901-e m p t y-v e c t o r组和S G C-7901-b l a n k组(均P<0.05);MTT法结果显示胃癌细胞GOLPH3基因过表达后,增殖能力明显增强,与SGC-7901-empty-vector组和SGC-7901-blank组相比差别有统计学意义(均P<0.05);SGC-7901-GOLPH3组、SGC-7901-empty-vector组和SGC-7901-blank组中mTOR总蛋白的表达无明显变化,而SGC-7901-GOLPH3组p-mTOR蛋白表达量较SGC-7901-empty-vector组和SGC-7901-blank组明显升高,与两对照组相比,上调GOLPH3能明显促进SGC-7901-GOLPH3组p-mTOR蛋白的表达(均P<0.05).结论:GOLPH3基因可能是通过上调mTOR信号通路中mTOR蛋白磷酸化水平来促进胃癌细胞增殖.
AIM： To observe the effect of GOLPH3 gene overexpression on the proliferation of gastric cancer cells. METHODS： The lentivirus containing the GOLPH3 gene was infected into gastric cancer cells, fluorescence microscopy was used to detect the expression of EGFP, and flow cytometry was applied to verify the infection efficiency. GOLPH3 overexpression was verified by quantitative realime PCR （at the mRNA level） and Western blot （at the protein level）. The expression of mTOR and p-mTOR proteins was detected by Western blot, and cell proliferation was assessed by MTT assay.RESULTS： Gastric cancer cells expressing high levels of EGFP were successfully obtained, with an infection rate of 100%. GOLPH3 expression was significantly up-regulated in SGC-7901 cells infected with the lentivirus containing the GOLPH3 gene compared with SGC-7901 ceils transduced with the empty vector or noninfected SGC-7901 cells （P 〈 0.05 for both）. MTT assay showed that up-regulated GOLPH3 could significantly enhance the proliferation of gastric cancer cells （P 〈 0.05）. Although the mTOR protein expression did not significantly differ between different groups of cells, but p-mTOR protein expression was enhanced in SGC-7901 cells infected with the lentivirus containing the GOLPH3 gene compared with control cells （P 〈 0.05）, suggesting that up-regulated GOLPH3 could promote the protein expression of p-mTOR in SGC-7901 cells. CONCLUSION： GOLPH3 gene overexpression could promote the proliferation of gastric cancer cells by up-regulating the expression of p-roTOR in SGC-7901 cells.
出处
《世界华人消化杂志》
CAS
北大核心
2014年第12期1669-1675,共7页
World Chinese Journal of Digestology
基金
广西科学研究与技术开发计划基金资助项目
No.桂科攻1140003A-13~~
