shRNA-FHIT对胃癌细胞株BGC-823增殖和凋亡的影响  

Effect of the short hairpin RNA of the fragile histidine triad on proliferation and apoptosis of the gastric cancer cell line BGC-823

作　　者：罗琼[1] 许洪伟[1] 郝菁华[1] 刘慧[1] 朱强[1] 

机构地区：[1]山东大学附属省立医院消化内科,济南250021

出　　处：《山东大学学报（医学版）》2010年第5期44-48,共5页Journal of Shandong University:Health Sciences

基　　金：山东省自然科学基金资助项目(2004ZX05)

摘　　要：目的构建靶向抑制脆性组氨酸三联体(FHIT)基因的短发夹双链RNA(shRNA)真核表达质粒,观察FHIT抑制后对胃癌细胞株BGC-823增殖和凋亡的影响。方法构建FHIT基因特异性的小RNA干扰质粒PGPU6/GFP/Neo-shRNA1、PGPU6/GFP/Neo-shRNA2,利用脂质体LipofactamineTM2000转染胃癌细胞BGC-823,实验分为未转染组、阴性对照组(转染PGPU6/GFP/Neo-shNC组)及PGPU6/GFP/Neo-shRNA1转染组和PGPU6/GFP/Neo-shRNA2转染组,G418筛选得到稳定表达株,Real-time PCR检测在mRNA水平干扰质粒对FHIT的抑制效应,MTT法、流式细胞术观察转染前后细胞生长特性的变化。结果与阴性对照组相比,转染shRNA-FHIT重组质粒的BGC-823细胞FHITmRNA表达明显下降(P<0.05)。与未转染组和阴性对照组相比,转染干扰质粒组细胞增殖活性增强,凋亡率减低、生长周期出现S期和G2/M期的比例上调(均P<0.05)。结论成功将重组shR-NA-FHIT表达载体转染BGC-823细胞,并筛选出稳定低表达FHIT的细胞。shRNA-FHIT可以促进胃癌细胞增殖、降低凋亡率,并削弱G0/G1期阻滞,为进一步研究FHIT基因在肿瘤中的作用机制奠定了实验基础。Objective To construct two eukaryotic plasmids expressing the short hairpin RNA（shRNA）of the fragile histidine triad（FHIT）and to study their effects on proliferation and apoptosis of the gastric cancer cell line BGC-823.Methods Specific shRNA plasmids to FHIT were constructed,and then transfected into the BGC-823 cells with lipofectamine methods.Cells were divided into four groups：the control group,the PGPU6/GFP/Neo-shNC transfected group as the negative group and the PGPU6/GFP/Neo-shRNA transfected groups.After selection with G418,stable cell clones were attained.Expression of FHIT mRNA was determined by quantitative real-time PCR.The effect of FHIT on the growth characteristics of gastric cancer cells was observed by methyl thiazolyl tetrazolium（MTT）and flow cytomery（FCM）.Results Stable clones with shRNA-FHIT plasmids were obtained.Compared with the negative control cells,expression of FHIT mRNA was down-regulated in the shRNA-FHIT plasmid transfected cells（P0.05）.Proliferation was promoted,whereas the cell apoptosis rate was decreased.Cells at the G0/G1 cell stage decreased,while the cells at the S and G2 cell stages increased.All these differences between shRNA-FHIT transfected cells and the two control groups of gastric cancer cells had statistical significances（P0.05）.Conclusions shRNA-FHIT plasmids were successfully transfected into BGC-823 cells,and the cells which expressed FHIT in a stable lower level were obtained.FHIT-targeted shRNA could obviously decrease the FHIT expression in BGC-823 cells at a stable lower level,promote the proliferation of BGC-823 cells,suppress their apoptosis and weaken the G0/G1 phase block of the cell cycle.

关 键 词：三联脆性组氨酸基因 胃肿瘤 RNA干扰 增殖 凋亡 

分 类 号：R735.2[医药卫生—肿瘤]