siRNA特异性抑制卵巢癌细胞葡萄糖调节蛋白78表达及其意义的研究  

Specific inhibition of Grp78 expression in ovarian carcinoma cell lines with RNA interference technique

作　　者：张丽颖[1] 徐爱丽[1] 吕楠[1] 李佩玲[1] 刘倩[1] 庄如锦[1] 原丹丹[1] 

机构地区：[1]哈尔滨医科大学附属第二医院妇产科,哈尔滨150086

出　　处：《中国优生与遗传杂志》2014年第7期14-17,23,共5页Chinese Journal of Birth Health & Heredity

基　　金：黑龙江省教育厅科技研究项目资助(1541145)

摘　　要：目的研究利用小分子干扰技术(RNAi),构建Grp78靶向RNA干扰质粒载体,观察其对人类卵巢癌细胞SKOV3Grp78基因表达的抑制作用。初步探讨RNA干扰技术抑制Grp78的表达作为卵巢癌基因治疗的可行性。方法从GeneBank中选取人类卵巢癌细胞Grp78基因序列,采用siRNA Target Designer-Version 1.51设计软件设计Grp78基因发卡寡核苷酸,序列符合siRNA表达载体psiSTRIKETMU6的要求并与psiSTRIKETM质粒载体连接,采用脂质体转染法将含有特异性小分子干扰Grp78 mRNA的重组载体psiSTRIKETM/Grp78导入卵巢癌细胞系SKOV3中,分别在转染前、转染后24h、48h和72h通过RT-PCR和Western blot检测Grp78 mRNA及蛋白水平的表达情况,用流式细胞仪检测细胞凋亡。四甲基偶氮唑蓝(MTT)比色法测定细胞经梯度浓度紫杉醇处理后的细胞存活率。结果成功构建RNA干扰质粒载体,靶向Grp78 RNA干扰质粒载体命名为psiSTRIKETM/Grp78。将上述质粒转染到卵巢癌细胞后,观察到psiSTRIKETM/Grp78能够有效的抑制Grp78 mRNA及蛋白表达。转染psiSTRIKETM/Grp78基因后的卵巢癌细胞增殖受到抑制,出现凋亡征象,且对化疗药物的敏感性增加。结论构建的RNA干扰真核表达载体psiSTRIKETM/Grp78能明显抑制Grp78 mRNA及蛋白的表达,增加卵巢癌细胞的化疗敏感性。RNAi技术抑制Grp78表达为卵巢癌的基因治疗开辟了新的思路。Objective： To construct of eukaryotic expression vector of RNA interference specific for Grp78 and observe silencing effect for Grp78 expression on human ovarian cancer cell line SKOV3. To explore the possibility and method of RNA interference technique inhibition Grp78 expression treating ovarian carcinoma. Methods： The gene sequences of human Grp78 were obtained from gene bank. A pair of Grp78 gene hairpin oligonucleotide was designed according to siRNA Target Designer-Version 1. 51 software which sequence meet the need of psiSTRIKETMu6 siRNA expression vector. Oligonucleotide designed coding expression of shRNA （small hairpin RNAs） for Grp78 gene were synthesized and inserted into plasmid psiSTRIKETM. Recombinant eukaryotic expression vector psiSTRJIZFJM/Grp78 containing RNA interference specific for Grp78 was introduced by lipofectamine transfection reagent into ovarian epithelial carcinoma cell line SKOV3o RT-PCR was used to detect the mRNA expression of Grp78 and the protein expression of Grp78 was detected by Western blot before transfection, 24h, 48h and 72h after transfection respectively. And cell apoptosis was assessed by flow cytometry. The cells proliferation and the viability rate after different concentrations of paclitaxel was assessed by methyl thiazolyl tetrazolium （MTF） . Results： The recombinant plasmid of RNA interference specific for Grp78 were successfully constructed and were named psiSTRIKETM/Grp78. Expression of Grp78 mRNA and protein were inhibited remarkablely by psiSTR1KETM/Grp78 after transfection. The cells proliferation was significantly inhibited after the transfection of psiSTRIKETM/Grp78 and showed the sign of apoptosis. The chemosensitity was enhanced. Conclusions： The eukaryotic expression vector psiSTRIKETM/Grp78 against Grp78 can significantly inhibit Grp78 expression in mRNA and protein. It can induce cell apoptosis and increase the sensitivity to Chemotherapy. And it provides evidence for gene therapy of human ovarian carcinomas.

关 键 词：RNA干扰(RNAI) GRP78 基因治疗 卵巢癌细胞 

分 类 号：R737.31[医药卫生—肿瘤]