RNA干扰技术体外抑制内质网分子伴侣GRP94在卵巢癌细胞表达的研究  

Specific inhibition of GRP94 expression in ovarian carcinoma cell lines with RNA interference technique

作　　者：张丽颖[1] 徐爱丽[1] 李佩玲[1] 艾丽敏 

机构地区：[1]哈尔滨医科大学附属第二医院妇产科 [2]哈尔滨市93220部队门诊部

出　　处：《中国优生与遗传杂志》2014年第10期16-20,共5页Chinese Journal of Birth Health & Heredity

基　　金：黑龙江省青年科学基金编号:QC2009C82;黑龙江省博士后科研启动金资助编号:LBH-Q11051;黑龙江省卫生厅科研课题编号:2007-320

摘　　要：目的利用RNA干扰技术,构建葡萄糖调节蛋白94(GRP94)靶向的RNA干扰质粒载体,观察其对卵巢癌细胞株HO-8910 GRP94基因表达的抑制作用及其对阿霉素(ADM)耐药的逆转作用。方法设计能转录小发卡结构RNA的DNA序列,并与psiSTRIKETM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,将重组质粒导入卵巢癌细胞株HO-8910内,实验分为3组,特异性siRNA组(HO-8910/siRNA组),非特异性siRNA组(HO-8910/NC siRNA组)和空白对照组(HO-8910组),用RT-PCR、Western blot和免疫荧光技术检测GRP94 mRNA及蛋白水平的表达情况,用流式细胞仪检测细胞凋亡,用四甲基偶氮唑蓝(MTT)法检测细胞对阿霉素化学敏感性的变化。结果成功构建RNA干扰质粒载体,转染针对GRP94的siRNA后24、48、72h,HO-8910的GRP94基因和蛋白水平明显下降;细胞凋亡增加;转染GRP94 siRNA后,HO-8910对阿霉素的敏感性显著增加。结论构建的RNA干扰真核表达载体能明显抑制GRP94 mRNA及蛋白的表达,促进细胞凋亡,通过降低卵巢癌细胞中GRP94的表达能增加其对阿霉素的敏感性,RNAi为卵巢癌的基因治疗提供了一种新策略。Objective： To construct of eukaryotic expression vector of RNA interference specific for GRP94 with RNA interference technique and observe silencing effect for GRP94 expression and reversing adriamycin （ADM） resistance in ovarian carcinoma cell lines. Methods： DNA designed coding expression of shRNA （small hairpin RNAs） for GRP94 gene was synthesized and inserted into plasmid psiSTRIKETM which is eukaryotic expression vector controlled by the U6 promoter of RNA polymerase Ⅲ, and cell line were transfected with recombinant plasmid by Lipofectamin 2000. The experimental cells were classified into HO-8910/siRNA, HO-8910/NC-siRNA and HO-8910. RT-PCR was used to detect the mRNA expression of GRP94 and the protein expression of GRP94 was detected by Western blot and immunofluorescence. Cell apoptosis was assessed by flow cytometry and the change of adriamycin sensitivity after interference were examined by methyl thiazolyl tetrazolium （MTT） assay. Results： The recombinant plasmid of RNA interference specific for GRP94 was successfully constructed. In HO-8910 cell lines, the expression of GRP94 mRNA and protein were dramatically decreased while the apoptosis ratio was increased 24, 48 and 72h after transfection. The sensitivity to adriamycin of HO-8910 was increased evidently after disturbing the GRP94 gene. Conclusions： The siRNA eukaryotic expression vector against GRP94 mRNA can significantly inhibit GRP94 expression, induce cell apoptosis, and increase the sensitivity to adriamycin of HO-8910 ceils. It provides evidence for gene therapy of human ovarian carcinomas.

关 键 词：卵巢癌 RNAI GRP94 

分 类 号：R737.31[医药卫生—肿瘤]