siRNA抑制肝癌细胞DNA修复门控基因表达的实验

Inhibition of Gatekeeper Genes of DNA Repair in Hepatoma Cell by Small Interfering RNA

目的本实验旨在研究siRNA抑制DNA修复门控基因Rad52、Ku70和Ku80的效果并筛选出高效的siRNA作用靶位。方法依据siRNA设计原则,针对每一个门控基因的mRNA序列各选择了2个靶位点并构建了相应的siRNA表达载体(psiRNA1～6)。酶切分析及DNA测序鉴定重组质粒构建成功后,将其转染人肝癌细胞株HepG2。RT-PCR和Western Blot分别用来检测psiRNAs在转录水平和翻译水平干扰靶基因的效果。结果psiRNA1～6作用细胞后明显抑制了靶基因的表达。比较而言,psiRNA1、psiRNA4、psiRNA5干扰效果分别比psiRNA2、psiRNA3、psiRNA6更佳。结论siRNA为进一步研究DNA修复门控基因Rad52、Ku70和Ku80的功能奠定基础。

Objective To study the inhibitory effects of small interfering RNA （siRNA） on expression of three gatekeeper genes （Rad52, Ku70 and Ku80） and screen the effective target sequences of siRNA. Methods According to the encoding sequences of gatekeeper genes, two pairs of oligonucleotide sequences targeted to every gatekeeper gene were designed and synthesized. The annealed oligonucleotide fragments were subcloned into psiRNA-hHneo expression vector. After being identified by restriction analysis and DNA sequencing, the recombinant plasmids （named as psiRNA1 ~6） were transfected into HepG2 cells. Inhibitory effects of siRNAs on the expression of three gatekeeper genes were examined by RT-PCR at RNA level and Western Blot at protein level, respectively. Results The expression of gatekeeper genes in transfected cells was down-regulated significantly by siRNAs. The plasmid-derived siRNAs by psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. Conclusion siRNA may provide us with practical tools for further study on the three gatekeeper genes.