人肺癌ILK基因siRNA重组表达质粒的构建及其对A549细胞增殖的影响

Construction of recombinant siRNA expression vector for ILK gene of human lung cancer and its effect on proliferation of A549 cells

目的构建人肺癌整合素连接激酶(Integrin-linked kinase,ILK)基因siRNA重组表达质粒,并检测其对人肺腺癌A549细胞增殖的影响。方法人工合成靶向ILK基因的siRNA干扰序列,克隆至载体pGenesil-1中,构建重组表达质粒pGenesil-1-ILK,利用脂质体转染A549细胞,经G418稳定筛选后,采用RT-PCR和Western blot检测细胞中ILK基因mRNA的转录水平及蛋白的表达水平,MTT法检测细胞的增殖活力。结果重组表达质粒pGenesil-1-ILK经SalⅠ单酶切及测序证明构建正确,其转染A549细胞后,细胞ILK基因mRNA的转录水平和蛋白的表达水平均明显降低(P<0.05),且细胞的增殖能力也显著降低(P<0.05)。结论成功构建了人ILK基因siRNA重组表达质粒,ILK基因沉默可显著抑制A549细胞增殖,为肺癌靶向基因治疗的研究提供了新的思路。

Objective To construct the recombinant siRNA expression vector for integrln-linked kinase （ILK） gene d human lung cancer and determine its effect on proliferation of A549 cells. Methods The siRNA sequence targeting to ILK gene was syn- thesized and cloned into vector pGenesil-1. The constructed recombinant plasmid pGenesil-l-ILK was transfected to A549 cells in mediation of liposome. Positive clones were screened with G418 and determined for transcription level of ILK mRNA by RT-PCR, ex- pression level of ILK protein by Western blot, and proliferation activity by MTT method. Results Recombinant siRNA expression vector pGenesil-l-ILK was constructed correctly as proved by digestion with Sac I and sequencing. After transfection with the recom- binant plasmid, the transcription level of ILK mRNA and expression level of ILK protein as well as the proliferation of A549 cells de- creased significantly （each P 〈 0. 05）. Conclusion The recombinant siRNA expression vector for ILK gene was constructed success- fully, and ILK gene silence inhibited the proliferation of A549 cells effectively, which provided a novel route for targeted gene thera- py of lung cancer.