shRNA干扰A549细胞KIAA0101基因表达的初步研究

Study on the expression of KIAA0101 gene in A549 cell by small hairpin RNA interference

目的探讨小发卡RNA(small hairpin RNA,shRNA)真核表达质粒介导的RNA干扰技术对人肺腺癌A549细胞KIAA0101基因表达的抑制作用。方法应用pSIREN-RetroQ载体构建KIAA0101基因shRNA重组质粒,经脂质体法导入A549细胞,分别设置为空白对照组、阴性对照组、干扰A组和干扰B组,采用实时定量PCR法和免疫印迹(Western blot)法检测检测转染后细胞KIAA0101基因mRNA与表达蛋白质的变化,四唑盐(MTT)比色法测定各组干扰细胞活性。结果成功构建KIAA0101基因shRNA重组质粒,相对阴性对照组与空白对照组,干扰B组KIAA0101的mRNA与蛋白质表达水平均下降达70%(P≤0.05)以上,MTT法结果显示降低KIAA0101的表达可抑制肺癌细胞的生长活性。结论shRNA干扰质粒可以显著降低细胞内KIAA0101mRNA与蛋白质的表达水平,并且抑制癌细胞的生长活性,为该基因在肺癌中的深入研究奠定了基础。

Objective To explore inhibitory effect of small hairpin RNA(shRNA)interference with eukaryotic expression plasmid on KIAA0101 gene expression in human A549 lung carcinoma cells.Methods KIAA0101 shRNA recombinant plasmid were constructed with pSIREN-RetroQ vector,and then were transfected into A549 cells with liposome.There were four groups:blank control group,negative control group,interference groups A and B.mRNA and protein expressions of KIAA0101 in the four groups were detected by qRT-PCR and Western bl...