| 王春梅,刘玉刚,白波.miR-29表达载体的构建及在HBV相关肝癌中靶基因的初步鉴定[J].济宁医学院学报,2012,(4):243-247 |
| miR-29表达载体的构建及在HBV相关肝癌中靶基因的初步鉴定 |
| Construction of miR-29 expression plasmid and preliminary identification of target in HBV-related HCC |
| 投稿时间：2012-06-03 |
| DOI：10.3969/j.issn.1000-9760.2012.04.004 |
| 中文关键词: microRNAs;miR-29;肝癌(HCC);乙型肝炎病毒(HBV);TUBB2A |
| 英文关键词: MicroRNA;Hepatocellular carcinoma (HCC);Hepatitis B virus (HBV);MiR-29;TUBB2A |
| 基金项目: |
| 作者 | 单位 | | 王春梅 | 济宁医学院泰山学者实验室, 山东济宁 272067 | | 刘玉刚 | 山东大学医学院病理生理学研究所, 山东济南 250012 | | 白波 | 济宁医学院泰山学者实验室, 山东济宁 272067 |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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