microRNA对冠心病疑似致病基因LPPR4表达调控的初步研究

发布时间：2018-03-11 17:12

  本文选题：早发冠心病　切入点：LPPR4　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：研究背景冠状动脉性心脏病简称冠心病(Coronary Artery Disease,CAD)在全世界范围造成严重的医疗和经济负担。男性发病年龄在55岁之前,女性在65岁之前的CAD称为早发冠心病(EarlyOnsetCoronary Artery Disease,EOCAD),遗传因素在 EOCAD发病中起到的作用尤为突出。我们收集了一个呈常染色体显性遗传的汉族早发冠心病家系,前期工作中通过全外显子组测序定位了 LPPR4基因3'UTR区单碱基缺失突变可能是该家系的致病基因,并发现该突变导致了 LPPR4蛋白表达下调。考虑到3'UTR区转录后表达调控与microRNA的关系,为了探究本家系患者早发冠心病的病理机制,需首先探明LPPR4基因表达是否通过miRNA进行调控。通过靶点预测网站检索发现候选变异正好位于miR-450a的识别位点中,此外既往文献报道miR-103a及miR-155-5p与冠心病密切相关,我们通过miRNA-DNA的靶效关系分析它们有可能与LPPR4 3'UTR区产生作用。研究目的了解目标3种miRNA与LPPR4基因蛋白表达及生理表型关系,明确目标miRNA是否与LPPR4基因3'UTR片段发生相互作用,初探后续调控表达相关机制。研究方法考虑到miRNA的效应与其在细胞内的表达丰度密切相关,首先行qPCR测定目标3种miRNA在包括血管平滑肌细胞在内的两种细胞系内的基线丰度。之后行干预实验,通过转染miRNA mimics后测定LPPR4蛋白表达量变化,以及行transwell试验测定细胞迁移变化,证实目标miRNA确与LPPR4表达调控相关。随后建立含有野生/突变型LPPR4 3'UTR区突变位点序列的荧光报告基因检测质粒,通过luciferase实验证明目标miRNA是通过与LPPR4基因3'UTR区前80位碱基序列结合而发挥起调控作用的。最后利用RNA聚合酶Ⅱ强抑制剂放线菌素D对细胞进行预处理抑制细胞转录,随即转染miRNAmimics,在不同时间点裂解细胞,行qPCR测定LPPR4 mRNA水平随时间推移的变化,以确定miRNA与LPPR4基因3'UTR区结合后是否通过影响mRNA稳定性实现调控。研究结果qPCR结果显示三种miRNA中miR-103a相对丰度最高。在miRNA干预实验中转染了 miR-103a及miR-450-5pmimics,行蛋白免疫印迹及transwell试验示高水平miR-450-5p对LPPR4基因的表达可能起下调作用,而miR-103a对LPPR4基因的表达可能起上调作用。双荧光素酶实验表明这两种miRNA与3'UTR区的确产生了相互作用,后续mRNA稳定性实验中miR-103a组LPPR4 mRNA降解率较对照组出现了下调,而miR-450-5p组和对照组间未发现显著差异。研究结论1.高水平miR-103a会上调LPPR4蛋白表达,并抑制血管平滑肌细胞的迁移;而miR-450-5p则下调LPPR4蛋白表达,并促进血管平滑肌细胞发生迁移。2.miR-450-5p及miR-103a均与LPPR4基因3'UTR区前80碱基序列存在相互作用。3.miR-450-5p与LPPR4基因mRNA稳定性调控相关。
[Abstract]:Background Coronary Artery distress (CAD) is a serious medical and economic burden worldwide. Women before the age of 65, CAD is called early onset coronary Artery disease, and genetic factors play a particularly important role in the pathogenesis of EOCAD. We collected an autosomal dominant Han family with early onset coronary heart disease. In the previous work, the single base deletion mutation in the LPPR4 gene 3UTR region was identified as the pathogenicity gene of the pedigree by whole exon sequence analysis. It was also found that the mutation resulted in down-regulation of LPPR4 protein expression. Considering the relationship between the regulation of post-transcriptional expression and microRNA in the 3-UTR region, the pathological mechanism of early onset coronary heart disease (CHD) was investigated in our family. It is necessary to find out whether the expression of LPPR4 gene is regulated by miRNA. By searching the target prediction website, we find that the candidate mutation is located in the identification site of miR-450a. In addition, it has been reported in previous literature that miR-103a and miR-155-5p are closely related to coronary heart disease. By analyzing the target activity relationship of miRNA-DNA, we analyzed their role in LPPR4 3 UTR region. Objective to understand the relationship between target 3 miRNA and LPPR4 gene protein expression and physiological phenotype, and to determine whether the target miRNA interacted with LPPR4 gene 3UTR fragment. The study considered that the effect of miRNA is closely related to its abundance in cells. The baseline abundance of target 3 miRNA in two cell lines, including vascular smooth muscle cells (VSMCs), was determined by qPCR. Then, the changes of LPPR4 protein expression after transfection of miRNA mimics and the changes of cell migration were measured by transwell assay. It was confirmed that the target miRNA was related to the regulation of LPPR4 expression. Subsequently, the fluorescent reporter gene detection plasmid containing the mutation site sequence of wild / mutant LPPR4 3 UTR region was established. It was proved by luciferase experiment that the target miRNA was regulated by binding to the first 80 bases of LPPR4 gene 3UTR region. Finally, actinomycin D, a strong inhibitor of RNA polymerase 鈪，

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