hnRNP K siRNA真核表达载体的构建及其在A549肺癌细胞中沉默效应的研究

Construction of eukaryotic expression vector of siRNA specific for hnRNP K and characterization of its efficiency in A549 lung carcinoma cell line

目的构建hnRNP K特异性siRNA真核表达载体,体外观察对hnRNP K基因的沉默作用。方法采用基因克隆技术,将合成的特异性hnRNP K RNA干扰寡核苷酸序列插入真核表达载体pSUPER,构建hnRNP K siRNA真核表达载体。采用Lipofect AMINE2000将pSUPER空载体和3个重组质粒(pSUPER/hnRNP K siRNAa,pSUPER/hnRNP K siRNAc和pSUPER/siRNAn)分别导入A549肺癌细胞(a、c、n分别代表hnRNP K编码序列中的A链和C链,以及无意义的对照序列Non链)。24 h后用RT-PCR和Western blot技术检测各实验组肺癌细胞内hnRNP K mRNA及蛋白水平的表达情况。结果成功构建了hnRNP K siRNA真核表达载体。转染hnRNP K siRNAa、hnRNP K siRNAc的肺癌细胞24 h后hnRNP K mRNA相对表达量分别为0.24±0.53和0.28±0.57,较对照组显著降低(P均<0.01);hnRNP K蛋白灰度值分别为0.23±0.11和0.28±0.09,较对照组显著降低(P均<0.05)。结论构建的RNA干扰真核表达载体能明显干扰A549细胞hnRNP K mRNA及蛋白的表达,为进一步研究hnRNP K基因的功能并应用于肺癌的治疗研究奠定了基础。

Objective To construct small interfering RNA （siRNA） eukaryotic expression vector specific for human hnRNP K gene, and to observe its silencing effects on hnRNP K gene in A549 cells. Methods The expression vectors of pSUPER/hnRNP K siRNAa, pSUPER/hnRNP K siRNAc and pSUPER/siRNAn were constructed by gene recombination and then transfected into the A549 lung carcinoma cell line by using Lipofect AMINE2000 （a and c respectively represented A and C fragments in hnRNP K coding sequence contained 19 nts, n represented nonsense fragment as control）. The mRNA and protein were harvested after 24 h and analyzed for the expression of hnRNP K by RT-PCR and Western blot respectively. Results The siRNA vector targeted to hnRNP K successfully decreased hnRNP K mRNA and protein levels 24 h after transfection in A549 cells. Relative expressed doses of hnRNP K mRNA in lung cancer ceils transfected by hnRNP K siRNAa and hnRNP K siRNAc respectively were 0. 24 ± 0. 53 and 0. 28 ± 0. 57 after 24 h, which were significantly lower than those in the control group （both P 〈 0. 01 ）. The gray scale values of hnRNP K protein were 0. 23 ± 0. 11 and 0. 28 ± 0. 09 respectively, which were also significantly lower than those in the control group （ both P 〈 0. 05 ）. And pSUPER/hnRNP K siRNAa was the most effective one. Conclusion Eukaryotic expression vector of siRNA specific for hnRNP K is successfully constructed,which lays the basis for the function study of hnRNP K gene and its application in the treatment of lung carcinoma.