摘要
目的检测串联siRNA对SW480细胞中增殖诱导配体（APRIL）基因的影响。方法设计并筛选针对APRIL基因的4条shRNA片段（sh644、shl451、shl938、sh2231），分别将其连接至pGenesil-1．1质粒表达载体，形成pGsh644、pGsh1451、pGshl938、pGsh2231单一表达载体，同时将4条shRNA连接至pGenesil4-T质粒形成pG4串联siRNA表达载体。阳离子脂质体法转染至结直肠癌细胞株SW480。实时荧光定量PCR和Westernblot技术检测SW480细胞中APRILmRNA和蛋白表达水平。结果经酶切和测序鉴定，构建的串联siRNA质粒载体符合要求。几条siRNA均可有效抑制SW480细胞APRIL基因的表达，APRILmRNA的抑制率分别是pGsh644（56．2％）、pGshl451（49．5％）、pGshl938（50．9％）、pGsh2231（49．2％）、pG4（79．3％）。蛋白质表达抑制率分别为pGsh644（56．9％）、pGshl451（48．7％）、pGshl938（50．1％）、pGsh2231（46．4％）、pG4（87．5％）（P〈0．05）。其中以pG4抑制效应最强。结论与传统单一的siRNA表达载体相比，串联siRNA表达载体大大地敲低了结直肠癌细胞株中APRIL基因的表达，为针对结直肠癌APRIL基因的基因治疗提供研究基础。
Objective To prove the remarkable inhibitive effect of multiple siRNAs targeting a proliferation-inducing ligand （APRIL） on the human colorectal cancer cell. Methods We constructed a multiple short hairpin RNA （shRNA） expression vector containing four shRNAs （pG4） as well as four single one （pGsh644, pGsh1451, pGsh1938, pGsh2231 ） against APRIL gene in SW480 cell, and then transfect- ed them into the human colorectal cancer cell line by cationic liposome. Ultimately, SW480 were screened by EGFP to obtain expression cell lines. APRIL expression levels including mRNA and APRIL protein were detected after transfected with all different kinds of vectors. Results A multiple shRNA expression vector containing four shRNAs （pG4） and four single ones were successfully constructed. Four single vectors （pGsh644, pGsh1451, pGsh1938, pGsh2231 ） and the multiple siRNAs expression vector （ pG4 ） all decreased the APRIL mRNA by 56.2% ,49.5% ;50.9% ,49.2% and 79.3%. And APRIL protein expres- sion was also remarkably reduced, especially by multiple siRNAs expression vector（87.5% ）. Conclusion Multiple siRNAs expression vector produced a more significant knockdown effect of APRIL than the vectors containing only one APRIL shRNA. What we found suggested us using the vector containing multiple shRNA to silence the expression of APRIL might be exploited as a novel therapeutic strategy for tumors.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2012年第7期629-633,共5页
Chinese Journal of Microbiology and Immunology
基金
江苏省医学重点学科项目（XK200723）
关键词
RNA干扰
串联siRNA
增殖诱导配体
结直肠癌
RNA interference （RNAi）
Multiple siRNA
A proliferation-inducing ligand
Coloncarcinoma
