小干扰RNA对胃癌细胞葡萄糖调节蛋白94表达的抑制作用  

Inhibitory effects of siRNA on expression of Grp94 in human gastric cell line

作　　者：张新晨[1] 杨维良[1] 徐华锋[2] 吴德全[1] 杨宝峰[3] 

机构地区：[1]哈尔滨医科大学附属第二医院普外科,150086 [2]黑龙江大学生命科学院 [3]哈尔滨医科大学药理教研室

出　　处：《中华实验外科杂志》2007年第5期543-545,641,共4页Chinese Journal of Experimental Surgery

基　　金：黑龙江省自然科学基金(D2004-23)

摘　　要：目的构建Grp94靶向RNA干扰质粒载体,观察其对人类胃癌细胞株SGC-7901 Grp94基因表达的抑制作用。方法从GenBank中选取人类Grp94基因序列,采用siRNA Target Designer-Version 1.51设计软件设计1对Grp94基因发卡寡核苷酸,序列符合siRNA表达载体siS- TRIKE^(TM)U6的要求并与psiSTRIKE^(TM)质粒载体连接,构建受控于人RNA聚合酶Ⅲ启动子U6的真核表达载体。采用Lipofectamne^(TM)2000转染试剂进行转染,将重组质粒导入人类胃癌细胞株SGC- 7901内,分别在转染前、转染后24、48和72h通过逆转录.聚合酶链反应(RT-PCR)、Western blot及免疫荧光技术检测Grp94 mRNA及蛋白水平的表达情况。结果成功构建RNA干扰质粒载体,靶向Grp94干扰质粒载体命名为psiSTRIKE^(TM)/Grp94,将上述质粒转染到人类胃癌细胞株后,观察到psiSTRIKE^(TM)/Grp94能够明显下调Grp94 mRNA及蛋白水平的表达,并随着时间推移稳定存在。结论构建的RNA干扰真核表达载体psiSTRIKE^(TM)/Grp94能明显抑制Grp94 mRNA及蛋白的表达,提示通过降低胃癌细胞中Grp94的表达,可能抑制或辅助抑制胃癌细胞的生长的作用。Objective To construct eukaryotic expression vector of RNA interference specific for Grp94 and observe silencing effect for Grp94 expression on human gastric cell llne SGC-7901. Methods The gene sequences of human Grp94 was obtained from gene bank. A couple of Grp78 gene hairpin oligonucleotide was designed,which sequence met the need of siSTRIKErU U6 siRNA expression vector. Oligonucleotides designed ceding expression of shRNA （small hairpin RNAs） for Grp94 gene were synthesized and inserted into plasmid psiSTRIKETM which was eukaryotic expression vector controlled by the U6 promoter of RNA polymerase Ⅲ, and human gastric ceil lines were transfected with recombinant plasmid by LipofectamneTM 2000. RT-PCR was used to detect the mRNA expression of Grp94 and the protein expression of Grp94 was detected by Western blot,immunofluorescence before transfection,24 h,48 h and 72 h after transfection respectively. Results The recombinant plasmids of RNA interference specific for Grp94 were successfully constructed and named psiSTRIKE^TM/Grp94 specific for Grp94. The expression of Grp94 mRNA and protein was suppressed significantly by psiSTRIKE^TM/Grp94 after ceil lines were transfected with recombinant plasmid. Conclusion The siRNA eukaryotic expression vector psiSTRIKE^TM/Grp94 against Grp94 mRNA can significantly inhibit the Grp94 expression. The results indicated that the Grp94 expression inhibited on human gastric cancer ceil line may inhibit growth of gastric cancer ceils.

关 键 词：胃癌 RNAI 

分 类 号：R735.2[医药卫生—肿瘤]