摘要
构建靶向ERCC6基因的短发夹RNA（short hairpin RNA,sh RNA）真核表达载体,评价其对肺癌细胞ERCC6表达的干扰效果。依据ERCC6基因的核苷酸序列和小干扰RNA的设计原则,设计并合成靶向ERCC6基因的3对ERCC6-sh RNAs和1对阴性对照NC-sh RNA片段,退火后连接至表达载体p GPU6/GFP/Neo中,重组载体经测序鉴定后转染肺癌SPC-A-1细胞中观察绿色荧光蛋白的表达情况,实时荧光定量PCR和Western blotting方法检测ERCC6基因表达的变化。经测序分析证实,各ERCC6-sh RNA的表达载体均构建成功。转染重组质粒48 h后的各组细胞均有绿色荧光表达。实时荧光定量PCR和Western blotting表明,相对于阴性对照组,ERCC6-sh RNA1、ERCC6-sh RNA2、ERCC6-sh RNA3三个实验组SPC-A-1细胞中ERCC6基因在m RNA和蛋白水平上的表达均显著降低（p〈0.05）,其中ERCC6-sh RNA3载体的干扰效果最佳。本研究成功构建并筛选了靶向ERCC6基因的sh RNA高效干扰载体,能够有效抑制ERCC6基因在肺癌细胞中的表达,这为进一步研究ERCC6基因与肿瘤发生分子机制和化疗耐药的相关性提供了帮助。
The aim of the study is to construct eukaryotic expression vectors of small hairpin RNA （shRNA） for the DNA repair gene ERCC6 and to evaluate the effect of ERCC6 knockdown in lung cancer cell. Three pairs of ERCC6-shRNAs and one pair of negative control NC-shRNA fragments were designed and synthesized according to the nucleotide sequence of ERCC6 and the criteria of designing small interfering RNA. Annealed shRNAs were ligated to the pGPU6/GFP/Neo vector. After shRNA recombinant vectors were vertified by the DNA sequencing, they were transfected into the SPC-A-1 cells. Green fluorescence protein was detected under microscope and the expression of ERCC6 gene was examined by Real-time PCR and Western blotting. Four shRNA vectors were successfully constructed by the sequencing analysis. Green fluorescence was detected in the cells transfected with recombinant plasmids for 48 h. Real-time PCR and Western blotting showed that the expression levels of ERCC6 mRNA and protein in SPC-A-1 cells transfected with ERCC6-shRNA vectors were significantly lower than those in SPC-A-1 cells transfected with negative control vector （p〈0.05）, and the ERCC6-shRNA3 vector inhibited the ERCC6 expression most efficiently. In conclusion, ERCC6-shRNA vectors were successfully constructed and screened in our study. ERCC6-shRNA vectors effectively inhibited the expression of ERCC6 gene in the lung cancer cells, which is helpful to further research the function of ERCC6 gene in tumorigenesis and chemotherapy resistance mechanism
作者
徐阳
胡芬
左桂福
王亮
郑向靖
孙渝祥
赵丽娜
Xu Yang;Hu Fen;Zuo Guifu;Wang Liang;Zheng Xiangjing;Sun Yuxiang;Zhao Lina(College of Life Science, North China University of Science and Technology, Tangshan, 063210;College of Material Seience and Engineering, North China University of Science and Technology, Tangshan, 063210)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2018年第7期3158-3163,共6页
Genomics and Applied Biology
基金
国家大学生创新创业训练计划项目(201510081010)
国家自然科学基金(No.51502075)
河北省高等学校科学技术研究项目(BJ2014027
QN20131059)
华北理工大学培育基金项目(GP201518)共同资助
