人miR-17-92基因簇载体构建及其在293T细胞中的表达验证

Construction of an Eukaryotic Expression Vector for Human miR-17-92 Cluster and the Validation of its Expression in 293T Cells

目的:研究人miR-17-92基因簇在肾癌等疾病发生过程中的功能。方法:采用基因重组技术,将miR-17-92基因簇亚克隆至pc DNA4真核细胞表达载体,构建miR-17-92基因簇真核表达载体;通过脂质体转染的方法将miR-17-92的过表达质粒和对照质粒分别转染293T细胞,并用实时定量PCR技术验证转染24和48 h后细胞内pri-miR-17-92和miR-17、miR-18a、miR-19a、miR-19b1、miR-20a、miR-92a1等分子的相对过表达比例。结果:miR-17-92基因簇真核表达载体构建成功,并在293T细胞中实现了显著性过表达效果。结论:为进一步研究人miR-17-92基因簇在肾脏发育过程中的功能及其与肾脏癌症发病相关性的研究提供了实验基础。

Objective： To explore the effects of miR-17-92 cluster in the pathogenesis of human renal cancer.Methods： Using the gene recombination technology, the gene fragments of miR-17-92 cluster were cloned from hu-man genomic DNA and inserted into the eukaryotic expression vector, pc DNA4 plasmid. The recombinant plasmidand its control vector were transfected into the 293 T cells respectively using Lipofect AMINE 2000, and the ex-pressed pri-miR-17-92 and the six mature miRNA members of miR-17-92 cluster（miR-17, miR-18 a, miR-19 a,miR-19b1, miR-20 a and miR-92a1） were detected using real-time quantitative PCR. Results： The constructs canbe used for overexpression of miR-17-92 cluster in 293 T cells. Conclusion： This work may provide useful toolsto clarify the functional roles of the miRNAs in human kidney development and to assess their potential associa-tions with the renal carcinogenesis in our coming studies.