本文選題：早發(fā)冠心病　切入點：LPPR4　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景冠狀動脈性心臟病簡稱冠心病(Coronary Artery Disease,CAD)在全世界范圍造成嚴重的醫(yī)療和經(jīng)濟負擔(dān)。男性發(fā)病年齡在55歲之前,女性在65歲之前的CAD稱為早發(fā)冠心病(EarlyOnsetCoronary Artery Disease,EOCAD),遺傳因素在 EOCAD發(fā)病中起到的作用尤為突出。我們收集了一個呈常染色體顯性遺傳的漢族早發(fā)冠心病家系,前期工作中通過全外顯子組測序定位了 LPPR4基因3'UTR區(qū)單堿基缺失突變可能是該家系的致病基因,并發(fā)現(xiàn)該突變導(dǎo)致了 LPPR4蛋白表達下調(diào)�？紤]到3'UTR區(qū)轉(zhuǎn)錄后表達調(diào)控與microRNA的關(guān)系,為了探究本家系患者早發(fā)冠心病的病理機制,需首先探明LPPR4基因表達是否通過miRNA進行調(diào)控。通過靶點預(yù)測網(wǎng)站檢索發(fā)現(xiàn)候選變異正好位于miR-450a的識別位點中,此外既往文獻報道m(xù)iR-103a及miR-155-5p與冠心病密切相關(guān),我們通過miRNA-DNA的靶效關(guān)系分析它們有可能與LPPR4 3'UTR區(qū)產(chǎn)生作用。研究目的了解目標(biāo)3種miRNA與LPPR4基因蛋白表達及生理表型關(guān)系,明確目標(biāo)miRNA是否與LPPR4基因3'UTR片段發(fā)生相互作用,初探后續(xù)調(diào)控表達相關(guān)機制。研究方法考慮到miRNA的效應(yīng)與其在細胞內(nèi)的表達豐度密切相關(guān),首先行qPCR測定目標(biāo)3種miRNA在包括血管平滑肌細胞在內(nèi)的兩種細胞系內(nèi)的基線豐度。之后行干預(yù)實驗,通過轉(zhuǎn)染miRNA mimics后測定LPPR4蛋白表達量變化,以及行transwell試驗測定細胞遷移變化,證實目標(biāo)miRNA確與LPPR4表達調(diào)控相關(guān)。隨后建立含有野生/突變型LPPR4 3'UTR區(qū)突變位點序列的熒光報告基因檢測質(zhì)粒,通過luciferase實驗證明目標(biāo)miRNA是通過與LPPR4基因3'UTR區(qū)前80位堿基序列結(jié)合而發(fā)揮起調(diào)控作用的。最后利用RNA聚合酶Ⅱ強抑制劑放線菌素D對細胞進行預(yù)處理抑制細胞轉(zhuǎn)錄,隨即轉(zhuǎn)染miRNAmimics,在不同時間點裂解細胞,行qPCR測定LPPR4 mRNA水平隨時間推移的變化,以確定miRNA與LPPR4基因3'UTR區(qū)結(jié)合后是否通過影響mRNA穩(wěn)定性實現(xiàn)調(diào)控。研究結(jié)果qPCR結(jié)果顯示三種miRNA中miR-103a相對豐度最高。在miRNA干預(yù)實驗中轉(zhuǎn)染了 miR-103a及miR-450-5pmimics,行蛋白免疫印跡及transwell試驗示高水平miR-450-5p對LPPR4基因的表達可能起下調(diào)作用,而miR-103a對LPPR4基因的表達可能起上調(diào)作用。雙熒光素酶實驗表明這兩種miRNA與3'UTR區(qū)的確產(chǎn)生了相互作用,后續(xù)mRNA穩(wěn)定性實驗中miR-103a組LPPR4 mRNA降解率較對照組出現(xiàn)了下調(diào),而miR-450-5p組和對照組間未發(fā)現(xiàn)顯著差異。研究結(jié)論1.高水平miR-103a會上調(diào)LPPR4蛋白表達,并抑制血管平滑肌細胞的遷移;而miR-450-5p則下調(diào)LPPR4蛋白表達,并促進血管平滑肌細胞發(fā)生遷移。2.miR-450-5p及miR-103a均與LPPR4基因3'UTR區(qū)前80堿基序列存在相互作用。3.miR-450-5p與LPPR4基因mRNA穩(wěn)定性調(diào)控相關(guān)。
[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，

本文編號：1599116