靶向黏着斑激酶siRNA表达载体构建及转染Tca8113细胞的沉默效应

Construction of the siRNA expression vector and the silencing influence on focal adhesion kinase gene of Tca8113 cell line

目的:构建靶向黏着斑激酶(focal adhesion kinase,FAK)基因的小干涉RNA(small interfering RNA,siRNA)质粒表达载体,转染人舌癌细胞Tca8113后观察其对FAK表达的抑制作用。方法:根据siRNA的设计原则,在人FAK mRNA序列中,设计并合成2条特异性靶序列寡核苷酸及2条无关对照序列,经退火成互补双链,再克隆到pGC-SilencerTM-U6/Neo真核表达载体中,构建重组体pSilencer-FAK并进行酶切鉴定;转染重组质粒至Tca8113细胞中,经G418抗性筛选,获得稳定转染细胞系;运用免疫细胞化学和Western-blot鉴定FAK的沉默效应。结果:质粒酶切证实目的寡核苷酸片段已被克隆到pGC-SilencerTM-U6/Neo载体中;pSi-lencer-FAK转染细胞后,FAK基因的表达受到明显抑制。结论:成功构建了针对人FAK的siRNA表达载体,通过转染Tca8113细胞可有效地抑制细胞中FAK的表达。

Objective-To construct the siRNA expression vector of focal adhesion kinase （FAK） gene and inhibit the expression of FAK gene in tongue cancer cell line Tca8113 by RNA interfering technique. Methods：According to the encoding sequence of FAK mRNA, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pGCSilencer^TM-U6/Neo siRNA expression vector, After being identified by restriction enzyme method, the recombinant pSilencer-FAK plasmids were transfected into Tca8113 cells. The transfected cells were selected by G418 method, Immuocytochemistry and Western blotting were used to evaluate FAK gene silencing efficiency, Results：The oligonucleotide fragments were correctly inserted into pGCSilencerTM-U6/Neo vector. FAK expression of the transfected cells was significantly down-regulated by pSilencer-FAK. Conclusion：The siRNA expression vector of FAK is successfully constructed and FAK expression of Tca8113 cells can be inhibited by RNA interfering technique.