【作者】 王晓；

【导师】 侯应龙；

【作者基本信息】 山东大学 ， 内科学（心血管病）， 2021， 硕士

【摘要】 研究背景心房颤动(房颤)是临床最常见的持续性快速性心律失常。房颤易引起血流动力学不稳定、血液高凝状态,易导致卒中、血栓栓塞、心功能不全等并发症,具有高致残率、高死亡率和治疗效果不佳的特点。研究表明,房颤使心衰的患病率增加3倍且加重心衰的症状,房颤患者心肌梗死的年发病率为0.4%-2.5%,其发生心肌梗死的风险较非房颤患者增加2倍。尽管房颤的药物治疗和介入治疗近年来取得了长足的进展,但目前使用的抗心律失常药物由于疗效不佳和药物自身的毒副作用而受到不同程度地限制,而有创导管消融术的并发症发生率虽然降低,但随着时间的推移,其房颤复发率仍然很高,这与房颤复杂的发病机制尚未完全阐明有关,因此,阐明房颤的发生机制,为房颤的精准治疗寻求干预靶点就显得尤为重要。众所周知,转录组即受上游基因组的调控,又受到组织分布、环境、疾病等因素的影响,并可以直接影响下游蛋白质的表达,故而在疾病的发生发展中起到重要作用。迄今,人类基因组计划测序工作已经完成,随着测序技术的不断发展,基因芯片技术作为有力工具已广泛应用于人类各种疾病的研究中。而从公共数据库中下载相关疾病的芯片数据,通过生物信息学分析,筛选差异表达基因并进行功能和通路富集分析,亦能为我们研究疾病发生发展的分子机制提供新的方法。研究目的本研究拟探究转录组在房颤发生中的作用和机制。研究分为两个部分,第一部分从GEO(Gene Expression Omnibus)数据库中检索房颤转录组数据集,通过生物信息学分析筛选与房颤相关的差异表达mRNA,然后通过构建细胞和动物模型来验证生信分析结果,确定目标mRNA。第二部分则是对目标mRNA进行GO功能和KEGG通路富集分析,预测目标mRNA可能的作用机制,然后通过体外功能获得实验,明确目标mRNA在房颤中发挥作用的具体机制,以期为房颤防治提供新的思路。研究方法1.下载房颤转录组相关的芯片数据并进行生物信息学分析在公共数据库平台检索、筛选并下载符合条件的转录组测序芯片数据,利用R语言中的limma包对芯片数据进行标准化和筛选差异表达mRNA,利用DAVID和String在线平台对差异表达mRNA进行GO、KEGG、PPI生物信息分析,筛选与房颤发生发展相关的差异表达mRNA。2.构建房颤原代心房肌细胞模型和房颤大鼠模型并验证生信分析结果分离并培养大乳鼠原代心房肌细胞,对其进行快速电场刺激(电压1.5V/cm,频率10Hz)。刺激72小时后提取RNA和蛋白,检测差异表达mRNA的表达变化。成年健康Wistar雄性大鼠共12只,随机分为2组:房颤组(n=6)和对照组(n=6);房颤组以固定频率(900bpm)进行高位右房快速起搏,连续起搏14天构建房颤大鼠模型,对照组行假手术后不进行起搏。14天后,两组分别取心房组织提取RNA和蛋白,检测差异表达mRNA的表达变化。3.预测目标mRNA的潜在作用机制利用DAVID在线分析平台对目标mRNA行GO功能和KEGG通路富集分析,筛查目标mRNA富集到的与房颤相关的生物功能和信号通路,寻找潜在的作用靶点,分析目标mRNA作用于房颤的分子机制。4.目标mRNA功能获得实验实验以人心房成纤维细胞(HCF)为研究对象,以外源性SPP1、PI3K抑制剂LY294002和丝裂霉素(MMC)为处理因素。将细胞随机分为对照组、SPP1处理组、MMC处理组、LY294002处理组、SPP1+MMC共处理组、SPP1+LY294002共处理组。处理24小时后提取蛋白质,应用Western Blotting法检测各指标的表达量变化。结果1.通过在GEO数据库中进行检索和筛选,最终有5个与房颤转录组相关的数据集被纳入本研究(GSE14975、GSE31821、GSE79768、GSE115574、GSE128188)。2.通过利用R语言软件中的limma包和RRA包对各芯片数据进行标准化、差异表达分析以及多数据集整合分析,从房颤-左心耳中筛选出147个差异表达mRNA,其中71个表达上调,76个表达下调。在房颤-右心耳中,共获得85个差异表达mRNA,其中34个上调mRNA,51个下调mRNA。3.GO功能富集分析表明差异表达的基因转录本主要参与了心传导调节、细胞外间隙和凋亡过程等生物学功能;KEGG通路分析主要富集到了钙离子信号通路上,富集到该通路上的差异表达mRNA有AGTR1、HTR2B、CD38、BDKRB1。利用Cytoscape软件,EGFR、ACTA1、SPP1、FHL2等被认为是蛋白质相互作用网络里的中心基因转录本。4.构建原代心房肌细胞房颤模型和房颤大鼠模型,对筛选出的差异表达mRNA进行验证,qRT-PCR和Western Blotting结果表明HTR2B和SPP1的表达变化趋势和生信分析结果一致,并选取SPP1作为目标mRNA行进一步深入的机制研究。5.SPP1功能获得实验证明,在细胞水平外源性增加SPP1可上调细胞纤维化相关基因的表达,包括collagen Ⅰ和α-SMA。通过对SPP1进一步的GO功能和KEGG通路分析,发现其参与细胞外基质、炎症反应等生物学过程并在PI3K/Akt/p27信号通路中发挥重要作用。6.与对照组相比,p27过表达可减弱SPP1所引起的促细胞纤维化作用,同时外源性增加SPP1可抑制p27的表达同时诱发Akt磷酸化;而PI3K抑制剂LY294002可阻断SPP1对下游的调控作用,表明SPP1通过PI3K/Akt信号通路调控p27的表达,进而发挥促房颤心房纤维化的作用。结论1.本研究通过跨数据集整合生物信息学分析,得出AGTR1、HTR2B、CD38、BDKRB1、EGFR、ACTA1、SPP1、FHL2、HPCAL4、APOA1、SPHKAP、LTBP1等mRNA可能参与房颤的发生发展,同时,钙离子信号通路可能在其中起到重要作用。2.细胞和动物水平实验证实SPP1和HTR2B的表达变化趋势与生信分析结果一致。3.SPP1可上调人心房成纤维细胞中的collagen Ⅰ和α-SMA的蛋白表达量,发挥促纤维化作用;并可以通过激活PI3K/Akt信号通路发挥对p27的抑制作用,上调collagen Ⅰ的表达,从而促进心肌细胞纤维化,参与房颤的发生发展。更多还原

【Abstract】 BackgroundAtrial fibrillation(AF)is the most common persistent tachyarrhythmia in clinical practice.AF is easy to cause hemodynamic instability,blood hypercoagulability,easy to lead to stroke,thromboembolism,cardiac dysfunction and other complications,and has the characteristics of high disability rate,high mortality and poor treatment effect.Studies have shown that AF increases the risk of heart failure by up to three times and exacerbates the symptoms of heart failure.Patients with AF have an annual incidence of myocardial infarction of between 0.4%and 2.5%,and their risk of developing myocardial infarction is two times higher than that of patients without AF.Although the drug and interventional treatment of AF has made great progress in recent years,the antiarrhythmic drugs currently used have different degrees of limitations due to poor efficacy and toxicity of the drug itself.While invasive catheter ablation has a lower complication rate,it still has a higher recurrence rate over time.This is related to the fact that the complex pathogenesis of AF has not been fully elucidated.Therefore,it is particularly important to clarify the pathogenesis of AF and seek intervention targets for the precise treatment of AF.It is well known that transcriptome is not only regulated by the upstream genome,but also affected by factors such as tissue distribution,environment and diseases,and can directly affect the expression of downstream proteins,so it plays an important role in the occurrence and development of diseases.Nowadays,the sequencing work of the Human Genome Project has been completed.With the continuous development of sequencing technology,gene chip technology has been widely used as a powerful tool in the study of various human diseases.The microarray data of related diseases were downloaded from public databases,and the differentially expressed genes were screened through bioinformatics analysis,and then the function and pathway enrichment analysis were performed.This provides a new method for us to study the molecular mechanism of disease occurrence and development.AimsThis study aims to explore the role and mechanism of transcriptome in the development of AF.The study consisted of two parts.In the first part,transcriptomic datasets were retrieved from the Gene Expression Omnibus(GEO)database,and bioinformatics analysis was performed to screen mRNAs associated with AF.Cell and animal models were constructed to verify the bioinformatic findings and the target mRNA was selected.In the second part,GO function and KEGG pathway enrichment analysis were performed on the target mRNA to predict its possible mechanism.The mechanism by which it plays a role in AF was further studied through in vitro functional acquisition experiments,in order to provide new ideas for the prevention and treatment of AF.Method1.Microarray data related to AF transcriptome were downloaded and analyzedTranscriptomic data sets were retrieved on the Gene Expression Omnibus(GEO)database,and bioinformatics analysis was performed to screen mRNAs associated with AF.Differential expression analysis was investigated with limma package in R software.Robudt Rank Aggreg package in R software was made use of constructing the integrate analysis.Gene Ontology(GO)annotations along with Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was performed on account of DAVID database(https://david.ncifcrf.gov/)and protein-protein interaction(PPI)analysis was performed on String online platform(https://string-db.org).2.Establishment of cell and animal models of AF and verification of bio informatics analysis resultsThe primary atrial myocytes were isolated from hearts of Wistar rats(1 day old)and stimulated by rapid electric field(10Hz,1.5V/cm).RNA and protein were extracted 72 hours after cell stimulation to detect the expression changes of differentially expressed mRNA.Twelve adult male Wistar rats weighing about 300-350g were randomly distributed to pacing group(n=6)and control group(n=6).In the pacing group,high right atrial rapid pacing was performed at fixed frequency(900bpm)for 14 consecutive days(6h/day)to establish the rat model of AF.The control group was not undergo pacing after sham operation.RNA and protein were extracted from atrial tissues of the two groups after 14 days,and the expression changes of differentially expressed mRNAs were detected.3.Prediction of potential mechanisms of target mRNAGo and KEGG analysis were performed on the target mRNA to screen the biological functions and signaling pathways related to AF.Further explore potential target sites and analyze the molecular mechanism of target mRNA acting on AF.4.Function acquisition experiment of target mRNAHuman atrial fibroblasts(HCF)were taken as the research object,and exogenous SPP1,PI3K/Akt pathway inhibitor LY294002 and mitomycin(MMC)were used as treatment factors.The cells were randomly divided into control group,SPP1 treatment group,MMC treatment group,LY294002 treatment group,SPP1+MMC co-treatment group,and SPP1+LY294002 co-treatment group.After 24 hours of treatment,proteins were extracted and the expression levels of relative proteins were detected by Western Blotting.Results1.Five AF-related transcriptome data sets(GSE14975,GSE31821,GSE79768,GSE115574,and GSE128188)were eventually included in this study.2.Limma package and RRA package in R software were used to conduct standardization,differential expression analysis and multi-dataset integration analysis of each chip data.As a result,147 differentially expressed mRNAs were screened from AF-left atrial appendage,of which 71 were up-regulated and 76 were down-regulated.In the AF-right atrial appendage,a total of 85 differentially expressed mRNAs were obtained,among which 34 mRNAs were up-regulated and 51 mRNAs were down-regulated.3.GO analysis showed that differentially expressed gene transcripts were mainly involved in cardiac conduction regulation,extracellular space and apoptosis.KEGG analysis was mainly concentrated in calcium signaling pathway,and the differentially expressed mRNAs concentrated in this pathway included AGTR1,HTR2B,CD38 and BDKRB1.EGFR,ACTA1,SPP1,and FHL2 were identified as central gene transcripts in the PPI analysis.4.The primary atrial myocytes AF model and rat AF model were established to verify the screened differentially expressed mRNAs.The results of qRT-PCR and Western Blotting showed that the expression trends of HTR2B and SPP1 were consistent with the results of bioinformatics analysis,and SPP1 was selected as the target mRNA for further mechanism study.5.The experiment proved that the exogenous increase of SPP1 at the cellular level could up-regulate the expression of fibrosis related genes,including collagen I and α-SMA.Through further analysis of GO and KEGG of SPP 1,it was found that SPP 1 is involved in extracellular matrix,inflammatory response and other biological processes,and plays an important role in PI3K/Akt/p27 signaling pathway.6.Compared with the control group,overexpression of p27 could reduce the pro-fibrosis effect induced by SPP1,while exogenous increase of SPP1 could inhibit the expression of p27 and induce Akt phosphorylation.However,the PI3K/Akt pathway inhibitor LY294002 can block the downstream regulation effect of SPP1,suggesting that SPP1 regulates the expression of p27 through the PI3K/Akt signaling pathway,and then plays a role in promoting atrial fibrosis in AF.Conclusion1.According to the integrated bioinformatic analysis of transcriptome sequencing data of AF,it is concluded that mRNA of AGTR1,HTR2B,CD38,BDKRB1,EGFR,ACTA1,SPP1,FHL2,HPCAL4,APOA1,SPHKAP and LTBP1 may be involved in the occurrence and development of AF.Meanwhile,calcium signaling pathway may play an important role.2.Cell and animal level experiments confirm that SPP1 and HTR2B expression trends are consistent with the results of bioinformatic analysis.3.SPP1 can up-regulate the protein expression of collagen I and α-SMA in HCF,and play a role in promoting fibrosis.Moreover,by activating the PI3K/Akt signaling pathway,SPP1 can play an inhibitory role on p27 and up-regulate the expression of collagen I,thereby promoting myocardial fibrosis and participating in the occurrence and development of AF.更多还原