双荧光素酶报告基因检测miR-147a与PDPK1靶标关系

发布时间：2018-04-11 07:18

本文选题：miR-147a + PDPK1　；参考：《新乡医学院》2017年硕士论文

【摘要】：背景:随着经济技术水平的不断提高,电离辐射已经完全充满了人们的日常生活。微小RNA(micro RNA,mi RNA)已经被发现在不同类型的辐射损伤中呈差异表达状态。有研究发现mi R-147a是放射损伤后的一个重要差异表达基因,它可以促使正常细胞发生凋亡导致辐射损伤的敏感性。PDPK1介导AKT的磷酸化,PI3K/PDPK1/AKT信号通路在辐射损伤的防护中起着重要作用,辐射后mi R-147a与PDPK1有相反关系。目的:研究mi R-147a与PDPK1之间的靶标关系,为下一步探讨mi R-147a和PDPK1基因在辐射致癌发展过程提供实验基础。方法:利用生物信息学软件Target Scan,mi Randa数据库,预测mi R-147a调控PDPK1基因结合序列。设计合成PDPK1 3'-UTR目的基因片段,将目的基因片段克隆到GV272荧光素酶报告基因载体,通过T4连接酶反应构建PDPK1 3'-UTR双荧光素酶报告基因野生型载体(GV272-PDPK1 3'-UTR)以及突变型载体(GV272-PDPK13'-UTR-mut);另外将mi R-147a PCR扩增片段和GV268荧光素酶报告基因载体相连,通过交换被交换反应构建GV268-mi R-147a重组载体。基因测序鉴定重组载体,验证重组载体构建成功。使用转染试剂Lipofectamine TM 2000 Reagent将这两种重组载体质粒共转染到293T细胞中,然后利用双荧光素酶基因检测系统检测其活性,以及利用western blot和荧光定量PCR验证二者相互关系,进而确定mi R-147a与PDPK1是否具有直接调控作用。结果:PCR基因测序结果表明,PCR扩增后含有PDPK1 3'-UTR和PDPK1 3'-UTR序列的片段的大小和序列与Gene Bank的报道序列一致。成功构建了两个含有mi R-147a结合位点的重组质粒及其突变重组质粒。双荧光素酶报告基因测定结果表明,与用PDPK1 3'-UTR突变载体质粒转染的mi R-147a组相比,荧光素酶活性与阴性对照组相比没有显着差异。通过转染PDPK1 3'-UTR载体,mi R-147a组荧光素酶的活性被明显抑制。荧光素酶活性与阴性对照组相比有显著性差异(P0.05)。在western、PCR、荧光显微镜验证PDPK1与mi R-147a的表达的实验中,mi R-147a组与阴性对照组相比表达量减少,具有显著性差异(P0.05);而加过抑制剂后,mi R-147a组表达量增多,具有显著性差异(P0.05)。结论:成功构建两组重组载体;PDPK1基因是mi R-147a的靶基因,mi R-147a结合到PDPK1基因的3'-UTR区域,mi R-147a和PDPK1具有负性调控表达,mi R-147a对转录后水平的PDPK1基因具有直接抑制作用。
[Abstract]:Background: with the development of economy and technology, ionizing radiation is full of people's daily life.Minute RNA(micro RNAi RNAs have been found to be differentially expressed in different types of radiation damage.It has been found that mi R-147a is an important differentially expressed gene after radiation injury. It can induce apoptosis of normal cells. PDPK1 mediates phosphorylation of AKT. PI3K / PDPK1 / AKT signaling pathway plays an important role in the protection of radiation injury.After radiation, mi R-147a has the opposite relationship with PDPK1.Aim: to study the target relationship between miR-147a and PDPK1, and to provide the experimental basis for the further study of the gene of mi R-147a and PDPK1 in the process of radiation carcinogenesis.Methods: Target Scanmi Randa database was used to predict the binding sequence of PDPK1 gene regulated by miR-147a.The target gene fragment of PDPK1 3'-UTR was designed and synthesized and cloned into GV272 luciferase reporter gene vector.Through T4 ligase reaction, PDPK1 3'-UTR double luciferase reporter gene wild-type vector, GV272-PDPK13, UTR-UTRand mutant vector, GV272-PDPK13-UTR-mutin, were constructed, and the PCR amplified fragment of mi R-147a was linked to GV268 luciferase reporter gene vector.GV268-mi R-147a recombinant vector was constructed by exchange exchange reaction.The recombinant vector was identified by gene sequencing, and the recombinant vector was successfully constructed.The recombinant plasmids were co-transfected into 293T cells with Lipofectamine TM 2000 Reagent, and their activity was detected by double luciferase gene detection system, and the relationship between them was verified by western blot and fluorescence quantitative PCR.Furthermore, it is determined whether mi R-147a and PDPK1 have direct regulation.Results the results of DNA sequencing showed that the size and sequence of the fragments containing PDPK1 3'-UTR and PDPK1 3'-UTR were consistent with the reported sequence of Gene Bank.Two recombinant plasmids containing mi R-147a binding site and their mutated recombinant plasmids were successfully constructed.The results of double luciferase reporter gene analysis showed that there was no significant difference in luciferase activity compared with the negative control group compared with the miR-147a group transfected with PDPK1 3'-UTR mutant plasmid.Luciferase activity was significantly inhibited by transfection of PDPK1 3'-UTR vector Mi R-147a.Luciferase activity was significantly different from that of negative control group (P 0.05).In the experiment of PDPK1 and mi R-147a expression verified by fluorescence microscope, the expression of PDPK1 and mi R-147a group was significantly lower than that of negative control group (P 0.05), but after adding inhibitor, the expression of miR-147a group was increased with significant difference (P 0.05).Conclusion: two groups of recombinant vector pPDPK1 gene were successfully constructed. The target gene of mi R-147a was mil R-147a binding to the 3'-UTR region of PDPK1 gene, and the expression of miR-147a and PDPK1 had direct inhibitory effect on PDPK1 gene at post-transcriptional level.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R818

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