串联siRNA对SW480细胞中增殖诱导配体基因表达的影响

Study on effects of tandem siRNA on proliferation-inducing ligand( APRIL) gene expression in SW480 cells

目的分析串联siRNA在SW480细胞中对增殖诱导配体(APRIL)基因的作用效果,探讨串联siRNA对APRIL基因表达的应用价值。方法设计筛选4条针对于APRIL基因的shRNA片段(即sh644、sh1938、sh1451、sh2231),分别将其与pGenesil-1.1质粒表达载体连接,形成4个单一表达载体(pGsh644、pGsh1938、pGsh1451、pGsh2231),同时将4条shRNA与pGenesil4-T质粒连接,共同构成pG4串联siRNA表达载体。应用阳离子脂质体法对结直肠癌细胞株SW480进行转染。采用实时荧光定量PCR以及Western blot法对SW480细胞的APRIL mRNA和蛋白表达进行检测。结果酶切及测序证实串联siRNA质粒载体构建成功。siRNA能够抑制SW480细胞APRIL基因表达,pG4组APRIL mRNA抑制率为77.72%,蛋白质表达抑制率为84.74%,显著高于其他质粒,差异具有统计学意义(P<0.05)。结论串联siRNA表达载体能够抑制结直肠癌SW480细胞株的APRIL基因表达,为结直肠癌APRIL基因治疗提供基础。

Objective To analyze the effects of tandem siRNA on proliferation-inducing ligand （APRIL） gene expression in SW480 cells and discuss its value. Methods We designed and selected 4 shRNA fragments for APRIL （ sh644, sh1938, sh1451, sh2231 ）, and they were connected with pGenesil-1.1 plasmid expression vectors, respectively. They formed 4 single expression vectors （pGsh644,pGsh1938,pGsh1451, pGsh2231） and meanwhile the 4 shRNA fragments were connected with pGenesil4-T plasmid for the constitution of pG4 tandem siRNA expression vector. We used cationic liposome method to transfect colorectal cancer SW480 cells. We used real-time quantitative PCR and Western blot method to detect APRIL mRNA and protein expressions in SW480 cells. Results Enzymes and sequencing test confirmed that the construction of siRNA plasmid vector series was successful. SiRNA can inhibit the APRIL gene expression in SW480 cells, APRIL mRNA inhibition rate was 77.72% in pG4 group. The inhibition rate of protein expression was 84.74%. It was significantly higher than other plasmids （P 〈 0.05 ）. Conclusion Tandem siRNA expression vector can inhibit the gene expression of APRIL in colorectal SW480 cells. It provides a basis for the treatment of APRIL gene of colorectal cancer.