小分子干扰RNA特异性抑制胃癌SGC7901细胞人端粒酶催化亚单位基因表达的研究  

Specific inhibition of hTERT gene expression by short interfering RNAs in gastric cancer SGC7901 cell

作　　者：马晋平[1] 詹文华[1] 汪建平[1] 彭俊生[1] 高劲松[2] 殷勤伟 

机构地区：[1]中山大学附属第一医院普通外科,广州510080 [2]中山大学达安基因诊断中心 [3]Division of Health Scienceand Technology Harvard Medical School

出　　处：《中华外科杂志》2004年第22期1372-1376,共5页Chinese Journal of Surgery

基　　金：国家自然科学基金 (3 0 2 712 76;3 0 2 714 69);广东省自然科学基金(0 2 1891)资助项目

摘　　要：目的　构建针对人端粒酶催化亚单位 (humantelomerasecatalyticsubunit,hTERT)基因的小分子干扰RNA(smallinterferingRNA ,siRNA)表达载体pU6 hTERT siRNAs,观察其对胃癌SGC790 1细胞hTERT基因的特异性抑制作用。方法　采用报告基因质粒pCX GFP( 5 5 10bp)和含有鼠U6启动子pU6 ( 330 0bp)质粒 ,设计合成针对绿色荧光蛋白 (GFP)基因的发夹RNA(shRNA)序列 ,构建SHi pU6 GFP质粒。应用LipofectamineTM2 0 0 0将pCX GFP质粒和SHi pU6 GFP质粒转染K5 6 2细胞、SGC790 1细胞 ,并用荧光显微镜观察转染效果。设计合成针对hTERT的siRNAs ,构建重组pU6 hTERT siRNAs质粒。LipofectamineTM2 0 0 0介导pU6 hTERT siRNAs质粒转染SGC790 1细胞。分别应用逆转录聚合酶链反应 (RT PCR)和荧光定量聚合酶链式反应 (FQ PCR)定性和定量检测hTERT基因表达情况。结果　质粒pU6和SHi pU6 GFP经EcoRⅤ和XbaⅠ酶切电泳后 ,前者出现 330 0bp和约 30 0bp条带 ,而后者出现 330 0bp和约 35 0bp条带 ,与实验设计的针对GFP的shRNA长度相符。说明本实验已成功构建了针对绿色荧光蛋白GFP基因的SHi pU6 GFP质粒。K5 6 2细胞和SGC790 1细胞中分别观察到转染pCX GFP质粒和SHi pU6 GFP质粒前后发绿色荧光的细胞数目明显减少。定性。Objective Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells. In present research, we investigated whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress human telomerase catalytice unit (hTERT) gene expression in gastric SGC7901 cells. Methods As a pilot study, we utilized green fluorescent protein (GFP) plasmid pCX-GFP(5 510 bp)as a reporter system and generated constructs SHi-pU6-GFP expressing small hairpin RNA (shRNA) specific for green fluorescence protein (GFP) in K562 and SGC7901 cell respectively. Furthermore, we constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene and transfected in SGC7901 by using Lipofectamine TM 2000. The expression of hTERT gene was detected by reverse transcription polymerase chain reaction(RT-PCR) and fluorescence quantitative polymerase chain reaction (FQ-PCR) assay. Results Our pilot study showed the short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into SGC7901 cell. The constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene was proved to be the same as designed by restriction endonuclease analysis. pU6-hTERT-siRNAs were successfully transferred into SGC7901 cell and their stable expression were obtained. The expression of hTERT gene were specific inhibited by pU6-hTERT-siRNAs in SGC7901 cell. Conclusions Short hairpin RNAs (shRNAs) could induce sequence-specific hTERT gene silencing in SGC7901 cell. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to cancer gene therapy.

关 键 词：SGC7901细胞 RNA 质粒 SH HTERT基因 转染 胃癌 绿色荧光蛋白(GFP) 小分子 GFP基因 

分 类 号：R735.2[医药卫生—肿瘤]