靶向胰腺癌PANC-1细胞整合素连接激酶基因RNA干扰质粒的构建及鉴定

目的:构建整合素连接激酶(integrin-linkedkinase,ILK)基因RNA干扰(RNA interference,RNAi)重组质粒并检测其对胰腺癌PANC-1细胞ILK基因表达的干扰效率,为进一步研究胰腺癌中ILK基因功能奠定实验基础和理论依据.方法:设计并构建3条含有针对ILK基因的小干扰RNA(small interfering RNA,siRNA)序列的重组质粒并进行DNA测序.通过阳离子脂质体Lipofectamine 2000将重组质粒转入人胰腺癌细胞株Panc-1细胞中,G418压力筛选至得到稳定转染细胞克隆,利用Real-Time PCR和Western blot检测ILK基因的表达抑制情况.筛选出干扰效率最高的重组质粒.结果:经DNA测序证实胰腺癌PANC-1细胞ILK基因的R NA干扰重组质粒构建成功;重组质粒稳定转染Panc-1细胞后(各组转染效率均>90%),各实验组ILK基因表达均被有效地抑制,其中重组质粒-2的干扰效率最高,其mRNA及ILK表达显著下调,其抑制率分别为93.01%和65.69%;ILK mRNA表达较阴性对照组、空质粒组表达量显著下降(0.090±0.009vs 1.147±0.110,1.005±0.121,P<0.01).结论:成功构建ILK基因RNA干扰重组质粒,重组质粒能有效抑制胰腺癌Panc-1细胞ILK基因表达.为进一步研究ILK在胰腺癌中的基因功能奠定基础.

Construction and identification of plasmids carrying small interfering RNAs targeting the ILK gene

AIM: To construct plasmids carrying small interfering RNAs(siRNAs) targeting the integrin-linked kinase(ILK) gene and assess their effect on ILK expression in pancreatic cancer cells. METHODS: Three pairs of siRNAs for ILK were designed and used to construct plasmids carrying siRNAs targeting the ILK gene.The recombinant plasmids and negative control plasmids were stably transfected into Panc-1 cells using cationic liposome Lipofectamine.After transfection,ILK mRNA and protein expression was detected by RT-PCR and Western blotting,respectively. RESULTS: DNA sequencing results indicated that the recombinant plasmids were constructed correctly.After stable transfection of the recombinant plasmids into Panc-1 cells,ILK mRNA and protein expression was significantly inhibited.Transfection of the recombinant plasmid that had the highest knockdown efficiency reduced ILK mRNA and protein expression by 93.01% and 65.69%,respectively.Compared to the non-transfected group and empty plasmid-transfected group,ILK mRNA expression was significantly down-regulated in the experimental group(0.090 ± 0.009 vs 1.147 ± 0.110,1.005 ± 0.121,both P < 0.01). CONCLUSION: Three plasmids carrying siRNAs targeting the ILK gene have been constructed successfully and provide a useful tool for studying the function of ILK.