摘要
目的通过构建miR-29的表达载体以及HBV相关肝癌中靶基因的初步鉴定,为探讨miR-29和其靶基因在HBV相关肝癌发生、发展中的作用奠定基础。方法利用生物信息学方法预测miR-29的靶基因,RT-PCR方法检测靶基因在肝癌细胞系及相应临床标本中的表达变化;构建miR-29及其靶基因的表达载体,利用双荧光报告检测方法检测miR-29与靶基因的靶向关系。结果成功构建miR-29及其靶基因核表达载体,分别命名为pS-miR-29a,pS-miR-29b,pS-miR-29c及pGL3-TUBB2A;靶基因TUBB2A在HBV相关肝癌中表达明显上调。结论 TUBB2A为miR-29的直接靶基因,本研究为下一步研究miR-29及其靶基因TUBB2A在HBV相关肝癌发生、发展中的作用奠定基础。
Objective MicroRNAs（miRNAs） are a class of non-coding,single-stranded RNA molecules and play important roles at the post-transcriptional level by imperfect base-pairing with 3’-untranslated regions（3’-UTR） of target mRNAs.In a preliminary study,we found that microRNA-29（miR-29） was significantly down-regulated in HBV-related HCC cell lines and HBV transgenic mice compared with their corresponding controls.Methods To further study the functions of miR-29,we amplified the precursors of human miR-29（miR-29a,miR-29b and miR-29c） gene by polymerase chain reaction（PCR） from the HepG2 genomic DNA,and then constructed miR-29 expression vectors pS-miR-29a,pS-miR-29b and pS-miR-29c.We combined the results from computational predictions,mRNA assays,gene chips and dual luciferase assays to identify the target of miR-29.Results The results indicated that miR-29c directly targeted TUBB2A（tubulin,beta 2A）,which were significantly upregulated in HepG2.2.15 cells compared with HepG2 cells as well as in HBV-related HCC tissue compared with NT.Conclusion The results would facilitate further studies of the functions of miR-29 and target genes during HBV infection and carcinogenesis of HCC.
出处
《济宁医学院学报》
2012年第4期243-247,共5页
Journal of Jining Medical University
