长链非编码RNA-032546、026102在心房颤动犬心脏内在自主神经重构中的功能及其机制研究

发布时间：2018-05-16 16:15

  本文选题：心房纤颤 + 心脏内在自主神经重构　； 参考：《山东大学》2016年博士论文

【摘要】：[研究背景]心房颤动(Atrial Fibrillation, AF,房颤)是临床上最常见的快速型紊乱性房性心律失常,发病率约0.4%-1%,且随年龄增长逐年增加。房颤的主要危害为栓子脱落引起的脑卒中及长期快速心室率诱发的心力衰竭,房颤患者死亡率是窦性心律人群的2倍。尽管药物、外科手术及导管射频消融等治疗房颤的手段在不断发展,房颤的治疗效果仍不尽人意。究其原因,与房颤发生机制不甚明了密切相关。继房颤的电重构及结构重构后,心脏内在自主神经重构,即心脏内在自主神经的再生过度与分布的不均一性,是房颤重要的发病机制。神经重构促进房颤发生与维持的同时,房颤的快速心房率也加剧心脏内在自主神经重构的发展,两者互为因果,形成恶性循环。阻止甚至逆转心脏内在自主神经重构可能是一种新型治疗房颤的方法。但目前对房颤神经重构现象的发生机制,仍知之甚少,亟需研究阐明。长链非编码RNAs (long non-coding RNAs,lncRNAs)是一类长度大于200个核苷酸,具有mRNA样结构,但缺少特异完整开放读码框架的转录本。与mRNAs相比,其表达在不同组织或同一组织的不同生长阶段具有明显的特异性,lncRNAs可从染色质重塑、转录调控及转录后加工等多种层面实现对基因表达的调控,被证实与蛋白质、DNAs和RNAs存在相互作用,因其特异性强和功能多样化,已成为现阶段研究的热点。现有研究表明,lncRNAs在心血管系统及神经系统的发育与发病过程中扮演重要的角色,lncRNAs不仅参与了神经元的分化、发育、突触可塑性及神经退行性疾病的发生、发展,且lncRNAs调控心脏疾病,如心肌梗死、心力衰竭、扩张型心肌病、室间隔缺损等的发病过程。以上均提示lncRNAs在房颤心脏内在自主神经重构的发生、发展过程中发挥重要生物学功能。但何种lncRNAs有异常表达,发挥何种作用,与房颤的发生、发展又有何种关联,尚无系统研究。[研究目的]通过快速右心房起搏建立房颤犬实验模型,应用高通量测序检测房颤及非房颤犬心房前右脂肪垫(anterior right fat pads, ARFPs)中差异lncRNAs的表达,并通过一系列生物信息学方法,鉴定与心脏内在自主神经重构相关的lncRNAs;通过体内、体外实验,研究lncRNAs对犬心脏内在自主神经重构及房颤诱发性的影响；并进一步探讨lncRNA-032546和lncRNA-026102影响房颤神经重构的机理,从lncRNAs的视角研究房颤神经重构发生的新机制,对房颤的防治提供新的干预靶点。[研究方法]1、房颤犬模型的建立取6只健康成年比格犬,雌雄不拘,随机分为对照组和起搏组。两组均放置起搏电极于右心耳,对照组不起博、喂养4周,起搏组以400次/min起搏4周建立房颤犬模型。为了检测房颤的发生,两组每周均至少行一次体表心电图。2、验证神经重构的发生取对照组及起搏组犬ARFPs组织,对神经细胞胞浆蛋白9.5(protein gene product 9.5, PGP 9.5)行免疫组化染色,计算神经密度,验证房颤介导神经重构的发生。3、高通量lncRNA测序并验证Triol法提取对照组及起搏组犬ARFPs中总RNAs,通过高通量二代测序检测lncRNAs及mRNAs的差异表达。并随机选取部分全新的lncRNAs,应用实时定量PCR (real-time quantitative PCR, qRT-PCR)的方法,对测序结果进行验证。4、筛选与房颤神经重构相关的lncRNAs对差异表达的转录本行Gene Ontology (GO)富集分析及Kyoto Encyclopedia of Genes and Genomes (KEGG)通路分析；通过差异性表达倍数、组织特异性、靶基因预测、文献检索等生物信息学方法,筛选并鉴定与神经重构相关的全新lncRNA-032546和lncRNA-026102。5、lncRNA功能缺失实验1)在体转染体外构建lncRNA-032546和lncRNA-026102的沉默慢病毒及对照慢病毒,滴度为1×109TU/ml。根据转染慢病毒的不同,15只比格犬随机分为空白转染组、低表达lncRNA-032546组和低表达lncRNA-026102组,开胸后将相应慢病毒在体斜行注射至ARFPs中,关胸,10天后处死,免疫荧光检测转染效率。2)电生理指标的检测通过程序电刺激分别于开胸后注射慢病毒前、注射慢病毒即刻及注射慢病毒后10天检测心房有效不应期(atrial effective refractory period, AERP)及房颤诱发率。检测区域为ARFPs周围0.5 cm内的心房肌。3)分子生物学指标的检测分别取转染组及对照组的ARFPs组织,对PGP 9.5行qRT-PCR及免疫组化染色,对神经生长因子,即生长蛋白相关43 (growth-associated protein 43, GAP 43)行qRT-PCR,计算神经密度,检测PGP 9.5及GAP 43 mRNAs的表达水平。6、lncRNAs潜在的作用机制1)Cis预测对lncRNAs进行分类,通过cis机制寻找IncRNAs上下游300 kb内的]mRNAs, qRT-PCR检测1ncRNAs及其上下游InRNAs的表达水平,明确其表达的相关性,结合文献进行分析。2) Trans预测根据皮尔逊相关系数及P值,计算并选取前100个与IncRNAs共表达的mRNAs,进行GO富集分析及KEGG通路分析,预测]lncRNA的trans作用机制。通过文献检索明确与IncRNAs有关的分子信号通路,进一步通过qRT-PCI测通路中关键分子的表达水平进行验证。[研究结果]1、通过快速起搏成功建立房颤犬实验模型,起搏组PGP 9.5阳性神经纤维的密度较对照组明显增加。2、通过二代高通量测序,共计获得61616个推定的IncRNAs,根据一系列筛选条件,得出1164个候选lncRNAs,其中,差异表达倍数2倍的有576个转录本,410个表达上调,166个表达下调,45个转录本被鉴定为全新的lncRNAs。随机选取6个全新的IncRNAs,qRT-PCR证实测序结果真实、可靠。3、GO富集分析及KEGG通路分析表明差异表达的基因主要参与了神经系统发育、细胞迁移及神经退行性疾病等生物学过程；通过筛选差异性表达倍数2倍的转录本、预测靶基因的生物学功能并去除在骨骼肌组织中非特异性高表达的lncRNAs,筛选出2个与神经重构相关的全新的表达下调的lncRNA-032546和lncRNA-026102。4、免疫荧光检测转染效率,qRT-PCR验证沉默效率；心内电生理结果表明,与对照组相比,AERP在转染前及转染即刻均无统计学差异,而转染后10天,lncRNA-032546表达下调可明显缩短AERP,相应的,短阵房速及房颤易诱发；与之相比,下调lncRNA-026102明显延长AERP,短阵房速及房颤不能诱发。5、PGP 9.5免疫组化结果表明,相比于对照组,低表达lncRNA-032546组神经密度明显增加,低表达lncRNA-026102组神经密度明显减少；qRT-PCR结果提示,GAP 43表达趋势与免疫组化结果类似,下调lncRNA-032546表达后,PGP9.5mRNA表达水平明显升高,而下调lncRNA-026102表达后,PGP 9.5的mRNA表达水平无统计学差异。6、lncRNA-032546和lncRNA-026102均为基因间IncRNAs,易通过cis机制调控邻近靶基因的表达。基于cis预测,找出lncRNA-032546和lncRNA-026102上下游300 kb内的mRNAs,qRT-PCR结果示下调lncRNA-032546和lncRNA-026102的表达分别上调CCND1-FGF19-FGF4-FGF3基因簇和SLC25A4的表达；通过文献检索明确差异表达的CCND 1-FGF 19-FGF4-FGF3基因簇和SLC25A4与神经生长、发育密切相关；进一步通过生物信息学计算lncRNA与mRNA间的共表达系数,推测出lncRNA-032546和lncRNA-026102潜在的靶基因分别为CCND1-FGF19-FGF4-FGF3基因簇和SLC25A4。7、根据trans预测,分别找出与lncRNA-032546、lncRNA-026102显著共表达的前100个mRNAs,行GO富集分析及KEGG通路分析。文献表明丝裂原活化蛋白激酶(MAPK)通路与CCND 1-FGF 19-FGF4-FGF3基因簇在促神经发生上密切相关,qRT-PCR结果表明ERK1、ERK2、JN及p38的mRNA表达水平均明显上调,提示下调lncRNA-032546的表达可激活MAPK通路发挥功能。同时,细胞核因子酉乙蛋白(NF-kappa B)通路在神经系统生长、发育中发挥重要作用,过表达SLC25A4通过招NF-kappa B诱导细胞凋亡,低表达lncRNA-026102组SLC25A4表达上调、神经细胞密度下调,提示下调lncRNA-026102介导神经重构的发生可能通过NF-kappa B通路上调SLC25A4的表达实现。[研究结论]1、在房颤／非房颤犬ARFP内一系列lncRNAs存在显著差异性表达,这些差异表达的lncRNAs参与了房颤心脏内在自主神经重构的发生；2、我们发现了2个全新的lncRNA-032546和lncRNA-026102与房颤神经重构密切相关,并通过功能缺失实验明确lncRNA-032546通过促进心脏内在自主神经的重构易化房颤的发生,而lncRNA-026102则抑制心脏内在自主神经重构进而阻止房颤的发生；3、我们预测了这2个lncRNAs的作用机制,即]lncRNA-032546通过cis调控邻近CCND1-FGF19-FGF4-FGF3基因簇并通过trans调控下游MAPK信号通路发挥作用；lncRNA-026102则通过c西调控邻近靶基因SLC25A4, trans调控TF-kappa B通路发挥生物学功能。[创新性]1、从lncRNAs的视角,观察其表达水平变化对房颤心脏内在自主神经重构的影响；2、探索lncRNAs通过cis调控邻近靶基因及trans调控下游分子信号通路影响房颤心脏内在自主神经重构的分子机制；3、应用犬心脏脂肪垫在体转染慢病毒介导的lncRNA沉默载体技术,研究lncRNAs对房颤心脏内在自主神经重构的影响及作用机制。
[Abstract]:Atrial fibrillation ( AF ) is one of the most common types of atrial fibrillation in patients with atrial fibrillation . AIM : To establish an experimental model of atrial fibrillation by rapid and right atrial pacing , and to identify the expression of different lncRNAs in anterior right fat pad ( ARFPs ) of atrial fibrillation and non - atrial fibrillation dogs by rapid and right atrial pacing .
In order to detect the occurrence of atrial fibrillation , two groups were randomly divided into two groups : control group and pacing group . In order to detect the occurrence of atrial fibrillation , two groups were randomly divided into control group and pacing group .
The expression levels of lncRNA - 032546 and lncRNA - 26102.5 and lncRNA - 26102.5 and lncRNA - 26102.5 and lncRNA - 26102.5 and lncRNA - 26102.5 , lncRNA - 032546 and GAP - 43 mRNAs were randomly divided into blank transfection group , low - expression lncRNA - 032546 group and low - expression lncRNA - 26102 group . Based on a series of screening conditions , 1,164 candidate lncRNAs were obtained , including 576 transcripts , 410 expression up - regulation , 166 expression downregulation , 45 transcripts were identified as completely new lncRNAs .
by screening transcripts with multiple times of differential expression , predicting the biological function of the target gene and removing the lncRNAs expressed in the non - specific high expression in the skeletal muscle tissue , two novel expression downregulated lncRNA - 032546 and lncRNA - 26102.4 related to nerve reconstruction are screened , the transfection efficiency of immunofluorescence detection is detected , and the silencing efficiency is verified by qRT - PCR ;
The results showed that the expression of lncRNA - 032546 could significantly shorten AERP after transfection .
Compared with the control group , the lower expression of lncRNA - 032546 group significantly increased the nerve density and the low expression of lncRNA - 032546 group was significantly decreased compared with the control group .
The results of qRT - PCR showed that the expression of GAP 43 was similar to that of immunohistochemistry . After downregulating the expression of lncRNA - 032546 , the mRNA expression level of PGP9.5 mRNA was significantly increased , but the expression of mRNA was regulated by cis mechanism . On the basis of cis prediction , the expression of lncRNA - 032546 and lncRNA - 26102 was found . The expression of lncRNA - 032546 and lncRNA - 26102 was downregulated and the expression of CCND1 - FGF19 - FGF4 - FGF3 gene cluster and SLC25A4 were regulated respectively .
The CCN1 - FGF - 19 - FGF4 - FGF3 gene cluster and SLC25A4 expressed by literature search were closely related to the growth and development of nerve ;
In this paper , we found that the potential target genes of lncRNA - 032546 and lncRNA - 26102 were significantly higher than that of CCND1 - FGF19 - FGF4 - FGF3 gene cluster and SLC25A4.7 .
2 . We have found that two new lncRNA - 032546 and lncRNA - 26102 are closely related to the remodeling of atrial fibrillation nerve , and through functional deletion experiments , lncRNA - 032546 is established to promote the reconstruction of the intrinsic autonomic nerve in the heart .
3 . We predict the mechanism of action of these two lncRNAs , that is , by cis - regulating the adjacent CCND1 - FGF19 - FGF4 - FGF3 gene cluster and regulating the MAPK signal pathway downstream through trans .
The effects of changes of expression level on the internal autonomic nerve remodeling in AF heart were observed from the perspective of lncRNAs from the perspective of lncRNAs .
2 . To explore the molecular mechanism of lncRNAs regulating the intrinsic autonomic nerve remodeling in patients with AF by regulating the molecular signaling pathway adjacent to the target gene and trans - regulating the downstream molecular signal pathway ;
3 . The effect and mechanism of lncRNAs on the internal autonomic nerve remodeling in patients with atrial fibrillation were studied by using canine cardiac fat pad in vivo .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.75
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本文编号：1897546