c-jun siRNA的构建及其对胃癌细胞株SGC7901中β3GnT8基因表达的影响

Construction,Screening of c-jun Effective siRNA Expression Vector and the Influence on β3GnT8 Gene Expression of SGC7901 Gastric Cells with c-jun Expression Blocked by RNA Interference

目的 构建c-jun干扰载体,有效沉默胃癌SGC7901细胞株中的c-jun基因表达,研究其对胃癌SGC7901细胞株中β3GnT8基因表达的影响.方法 ①利用互联网资源针对c-jun基因的靶序列设计合成四条siRNA,将四条siRNA插入到RNA干扰载体pGPU6/GFP/Neo中构建成四条干扰载体.②将构建好的c-jun siRNA载体质粒pGPU6/GFP/Neo-sic-jun-1949、pGPU6/GFP/Neo-sic-jun-1050、pGPU6/GFP/Neo-sic-jun-1111、pGPU6/GFP/Neo-sic-jun-1276通过阳离子脂质体将表达质粒分别转染SGC7901细胞,48h后荧光显微镜下观察不同浓度质粒的转染效率,通过RT-PCR法检测c-jun和β3GnT8基因mRNA的表达.结果 ①成功构建了四条正确的c-jun siRNA表达质粒;②成功筛选出一条对c-jun有明显抑制作用的siRNA干扰质粒,阻断c-jun表达后,糖基转移酶β3GnT8的表达受到明显抑制.结论 构建的c-jun siRNA表达质粒可以有效抑制胃癌SGC7901细胞株的c-jun基因表达,同时抑制糖基转移酶β3GnT8的表达,为研究胃癌的分子机制提供了一定的理论基础.

Objective To construct a vector of RNA interference（RNAi） on c-jun and to effectively silence the gene expression of [3 3GnT8 in SGC7901 gastric cells.Methods （1）Four possible small interfering RNA（RNAi） was designed for c-jun gene mRNA through the use of Internet resources.These four small interfering RNA were recombined with the vector pGPU6/GFP/Neo,and four vectors of RNA interference were built.（2）SGC7901 gastric cells were transfected with the constructed vector pGPU6/GFP/Neo-shc-jun-1949,pGPU6/GFP/Neo-shc-jun- 1050,pGPU6/GFP/Neo-shc-jun- 1111,pGPU6/GFP/Neo-shc-jun- 1276 plasmid,and the transfection efficiency could be observed by the Fluorescence microscope of those transfected with different concentrations of plasmid.The expression variation of bone-related gene such as c-jun and β 3GriT8 were detected by RT-PCR.Results （1）Four correct c-jun expression vector of RNAi were constructed successfully.（2）The siRNA expression vector that could block the c-jun effectively was screened out.The β 3GnT8 gene mRNA were all inhibited distinctly after the gene c-jun was blocked.Conclusion The constructed c-jun siRNA expression vector could effectively inhibit the c-jun gene expression of SGC7901 gastric cells,and could also inhibit the expression of β 3GnT8 gene.The β 3GnT8 study has laid an experimental foundation for the therapy of gastric cells.