靶向人肝素酶短发卡状RNA表达载体的构建及其沉默作用  

Construction of heparanase-targeted shRNA expression vector and its gene silencing effects

作　　者：蒋国松[1] 曾甫清[1] 郑丽端[2] 童强松[1] 董继华[3] 侯晓华[4] 

机构地区：[1]华中科技大学同济医学院附属协和医院外科,武汉430022 [2]华中科技大学同济医学院附属协和医院病理科,武汉430022 [3]华中科技大学同济医学院附属协和医院中心实验室,武汉430022 [4]华中科技大学同济医学院附属协和医院消化内科,武汉430022

出　　处：《中华实验外科杂志》2010年第5期623-625,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30200284,30600278,30772359）;教育部“新世纪优秀人才支持计划”资助项目（NCET-06-0641）;教育部回国人员基金资助项目（2008889）

摘　　要：目的构建靶向人肝素酶（HPSE）的短发卡状RNA（shRNA）表达载体，探讨其基因沉默作用。方法根据人肝素酶mRNA序列设计shRNA，合成两条互补的寡核苷酸链，退火后连接人pGenesil-1载体，转化扩增后进行测序鉴定；重组质粒在阳离子脂质体Lipofectamine2000的介导下，瞬时转染人胃癌细胞株SGC-7901、人前列腺癌细胞株PC-3、人膀胱癌细胞株EJ，每种细胞分别设空白对照组、空载体pGenesil-Negative转染组、pGenesil-HPSEshRNA转染组3组，逆转录-聚合酶链反应（RT-PCR）及Westernblot法检测各种细胞中肝素酶基因表达水平，黏附实验和transwell小室实验检测癌细胞游走、侵袭能力。结果所构建的shRNA载体插入基因片段与设计序列完全匹配，载体构建成功；重组质粒转染SGC-7901、PC-3、EJ细胞后，肝素酶mRNA表达分别下调78．6％（P〈0．01）、82．6％（P〈0．01）、81．9％（P〈0．01），肝素酶蛋白表达分别下调76．4％（P〈0．01）、85．9％（P〈0．01）、83．3％（P〈0．01），细胞黏附能力分别下降37．7％（P〈0．01）、44．6％（P〈0．01）、43．8％（P〈0．01），细胞侵袭能力分别下降66．8％（P〈0．01）、76．6％（P〈0．01）、80．5％（P〈0．01）。结论成功构建了靶向人肝素酶的shRNA表达载体，沉默肝素酶基因表达后，能抑制多种癌细胞的游走和侵袭能力。Objective To construct heparanase-targeted short hairpin RNA （shRNA） expression vector and investigate its gene silencing effects. Methods Heparanase-targeted shRNA was designed. Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector, which was validated by nucleic acid sequencing. Under the induction of Lipofectamine 2000, the recombinant was transferred into SGC-7901, PC-3 and EJ. Each cell line was divided into three groups： the control group, the pGenesil-Negative group, and the pGenesil-HPSE shRNA group. The expression levels of heparanase were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. The cell adhesion and transwell-ECM assays were applied for the detection of in vitro migration and invasion of canc- er ceils. Results Sequencing analysis confirmed the correctness of the construction. Compared with nega- tive control, the expression of heparanase mRNA was downregnlated by 78.6% （ P 〈 0.01 ） , 82.6% （P 〈0.01 ） , 81.9% （P 〈0.01 ） and the expression of heparanase protein was downregnlated by 76.4% （P〈0.01）, 85.9% （P〈0.01）, 83.3% （P〈0.01）, respectively afterSGC-7901, PC-3 andEJcells were tranfected with the recombinant. Meanwhile, the cellular adhesion was downregnlated by 37.7% （P 〈0.01 ） , 44.6% （P 〈0.01 ） , 43.8% （P 〈0.01 ） , and the invasive activity was downregulated by 66.8% （P〈0.01）, 76.6% （P〈0.01）, 80.5% （P〈0.01）, respectively. Conclusion The heparanase-targeted shRNA expression vector was successfully constructed, and reduced the gene expression of heparanase in tumor ceils, resulting in the decrease of migration and invasion of cancer cells, which provided a novel strategy for ameliorating the malignant biological characteristics of tumor.

关 键 词：肝素酶 短发卡状RNA 肿瘤 基因表达 

分 类 号：R730.2[医药卫生—肿瘤]