人CD2相关蛋白剪接异构体启动子区的克隆及microRNA-548k对其调控机制的研究

发布时间：2018-06-01 21:04

  本文选题：CD2相关蛋白 + 转录调控　； 参考：《南京医科大学》2017年硕士论文

【摘要】：目的:克隆人CD2相关蛋白剪接异构体(CD2AP002)启动子区,探讨microRNA-548k(miR-548k)能否通过靶向结合该剪接异构体启动子序列参与其转录调控。方法:采用PCR方法获取CD2AP002基因5'侧翼序列及一系列5'端缺失体,定向插入到pGL3-Basic载体中,双萤光素酶报告基因检测系统鉴定其启动子活性;RegRNA2.0在线软件预测CD2AP002启动子区域潜在的miR-548k的结合位点;荧光原位杂交实验(fluorescence in situ hybridization,FISH)检测 miR-548k 在HEK293细胞内的定位,细胞免疫荧光实验(immunofluorescence staining,IF)观察RNA干扰蛋白(AG02)在HEK293细胞内的分布;利用点突变/缺失突变技术、miR-548k 模拟剂(miR-548k mimic)/miR-548k 抑制剂(miR-548k inhibitor)的转染、实时定量PCR(Real time quantitative PCR)技术,鉴定miR-548k是否能通过靶向CD2AP002启动子的潜在结合位点调控CD2AP002的转录;转染miR-548k 模拟剂(miR-548k mimic)后,染色质免疫沉淀(chromatin immunoprecitation,ChIP)检测RNA聚合酶Ⅱ在人CD2AP002启动子区域的富集情况。结果:成功构建有活性的人CD2AP002质粒;通过生物信息学预测,在CD2AP002启动子区域含有两个miR-548k靶向结合位点;FISH证实miR-548k可见于HEK293细胞核、IF证实AG02蛋白在HEK293细胞的胞核里有分布;miR-548k通过结合CD2AP002启动子上两个结合位点可在启动子和mRNA水平上正向调控CD2AP002;ChIP实验发现RNA聚合酶Ⅱ在CD2AP002启动子区的富集。结论:成功构建出有活性的人CD2AP002质粒;miR-548k通过靶向结合CD2AP002启动子上调CD2AP002的表达,该过程中有RNA聚合酶Ⅱ的富集。
[Abstract]:Aim: to clone the promoter region of human CD2 related protein splicing isomer CD2AP002, and to investigate whether microRNA-548k- miR-548k) can participate in its transcriptional regulation by targeting the splicing isomer promoter sequence. Methods: the 5'flanking sequence of CD2AP002 gene and a series of 5'terminal deletions were obtained by PCR and inserted into pGL3-Basic vector. The double luciferase reporter gene detection system identified its promoter activity, RegRNA2.0 online software for predicting potential miR-548k binding sites in the CD2AP002 promoter region, fluorescence in situ hybridization assay (fish) for the localization of miR-548k in HEK293 cells, and fluorescence in situ hybridization assay (fish) for detecting the localization of miR-548k in HEK293 cells. The distribution of RNA interference protein (AG02) in HEK293 cells was observed by immunofluorescence assay, the transfection of miR-548k mimic agent miR-548k by point mutation / deletion mutation technique, and the real-time quantification of PCR(Real time quantitative PCR) were performed. To determine whether miR-548k can regulate the transcription of CD2AP002 by targeting the potential binding site of CD2AP002 promoter, and to detect the enrichment of RNA polymerase 鈪，

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