【作者】 王静；

【导师】 林静娜；

【作者基本信息】 天津医科大学 ， 临床医学（专业学位）， 2023， 博士

【摘要】 背景:急性心肌梗死(acute myocardial infarction,AMI)是全球主要死亡原因之一,缺氧/再复氧(hypoxia/reoxygenafion,H/R)损伤是AMI的关键病理过程,可引起心肌细胞氧化应激和炎症损伤,最终导致心肌细胞更进一步的凋亡和坏死。环状RNA(circular RNA,circRNA)是一类由前体mRNA成熟时外显子或内含子反向剪切(back splice)形成的单链、闭合RNA。研究表明,circRNA可以通过miRNA海绵作用,干扰miRNA的功能,从而影响心血管疾病的发生和发展。目前关于circRNA在心肌细胞H/R损伤中的作用有待进一步的研究和探索。目的:本研究旨在探讨AMI相关的circRNA,并研究circUSP39在H/R介导的心肌细胞损伤中发挥的作用,并进一步探讨circUSP39调控H/R介导的心肌细胞损伤的潜在分子机制。方法:1.我们通过分析GEO数据库中AMI相关的circRNA芯片数据,筛选AMI中差异表达的circRNA,另通过缺氧/再复氧处理人心肌细胞株AC16细胞建立H/R心肌细胞损伤模型,qRT-PCR检测差异表达的候选基因,circUSP39的表达明显升高。然后,进行CCK8实验、酶联免疫吸附实验和流式细胞术评估circUSP39功能缺失对H/R引起的心肌细胞增殖、氧化应激、炎症反应和细胞凋亡的影响。2.探讨circUSP39调控H/R心肌细胞损伤的分子机制。首先利用在线生物学数据库分析与circUSP39具有结合位点的miRNAs。应用Ago2 RIP、RNA pull down实验和双荧光素酶报告基因实验进行验证。通过在线生物信息学数据库star Base分析与miRNA具有靶向结合位点的下游基因。另在心肌细胞株中构建过表达载体,通过细胞功能实验探索作用机制。3.对上述发现进行临床验证。急性ST段抬高型心肌梗死(st-elevated myocardial infarction,STEMI)行急诊介入治疗患者为试验组,同期行冠脉造影检查的非介入治疗患者为对照组,每组各10例患者入组,检测两组circUSP39、下游目标基因、氧化应激及炎症指标的水平。结果:1.circUSP39在AMI患者血液样本及H/R诱导的心肌细胞中均显著高表达。与对照组比较,H/R处理后,心肌细胞的增殖能力降低,细胞氧化应激水平和炎症反应水平升高,细胞凋亡率升高。与H/R+si-NC组(阴性对照组)比较,circUSP39敲减组心肌细胞的增殖能力增强,细胞氧化应激水平和炎症反应水平降低,细胞凋亡率降低。2.Ago2 RIP、RNA pull down实验和双荧光素酶报告基因实验证实circUSP39直接靶向结合miR-362-3p。细胞功能拯救实验结果显示,抑制miR-362-3p后,circUSP39敲低对H/R引起的心肌细胞氧化应激、炎症反应和细胞凋亡的抑制作用均减弱,细胞增殖能力降低。另star Base预测到miR-362-3p与TRAF3的3’UTR具有互补结合位点。qRT-PCR和western blot结果显示,H/R诱导的心肌细胞中TRAF3 mRNA和蛋白水平均呈高表达。双荧光素酶报告基因实验证实miR-362-3p能够与TRAF3的3’UTR靶向结合。qRT-PCR和western blot结果表明,在心肌细胞株AC16中,过表达miR-362-3p可抑制细胞中TRAF3mRNA和蛋白表达;而抑制miR-362-3p则可上调细胞中TRAF3 mRNA和蛋白表达。细胞功能实验结果显示,过表达miR-362-3p能够增强H/R诱导的心肌细胞增殖,抑制H/R引起的细胞氧化应激、炎症反应和细胞凋亡;而同时过表达TRAF3则能逆转这种作用。此外,通过qRT-PCR和western blot实验检测到,在心肌细胞株AC16中,circUSP39可通过靶向miR-362-3p正向调控TRAF3基因表达。3.在临床实验部分,与对照组相比,试验组circUSP39、TRAF3、氧化应激及炎症指标明显升高,具有统计学差异;miR-362-3p水平明显下降,具有统计学差异。结论:1.H/R诱导心肌细胞增殖能力降低和细胞凋亡增加,并刺激细胞发生氧化应激和炎症反应,circUSP39促进上述反应;而敲低circUSP39能够抑制H/R介导的心肌细胞增殖降低,减轻细胞氧化应激和炎症反应水平,并抑制细胞凋亡。2.circUSP39能够直接靶向miR-362-3p,而TRAF3是miR-362-3p的一个靶基因。circUSP39通过靶向miR-362-3p/TRAF3轴调控心肌细胞H/R损伤。揭示circUSP39/miR-362-3p/TRAF3轴可能是心肌细胞H/R损伤过程中的一个新机制,这一结果可能为改善AMI预后提供潜在的治疗靶点。3.本研究通过临床实验证实,氧化应激及炎症因子参与H/R损伤,circUSP39/miR-362-3p/TRAF3轴在AMI患者中存在表达差异,参与H/R损伤过程,与细胞实验观察到结果的一致。更多还原

【Abstract】 Background:Acute myocardial infarction(AMI)is one of the leading causes of death worldwide,hypoxia/reoxygenafion(H/R)injury is a key pathological process after AMI,which can cause oxidative stress and inflammatory damage of cells,and eventually lead to further apoptosis and necrosis of cardiomyocytes.Circular RNA(circRNA)is a type of single-stranded,closed RNA formed by exon or introns reverse shear(back splice)of precursor mRNA at maturation.These studies have shown that circRNA can interfere with the function of miRNA through miRNA sponge,thus affecting the development of cardiovascular disease.Further research and exploration are currently needed regarding the role of circRNA in myocardial H/R injury.Purpose:This study aimed to investigate AMI-related circRNA and investigate the role that circUSP39 plays in H/R-mediated cardiomyocyte injury and to further explore the underlying molecular mechanisms of circUSP39 regulating H/R-mediated cardiomyocyte injury.Methods:1.We screened the differentially expressed circRNA in AMI by analyzing AMI-related circRNA microarray data in the GEO database,in addition,an H/R cardiomyocyte injury model was established by hypoxia/reoxygenation treatment of human myocardial cell line AC16 cells,and the differentially expressed candidate genes were detected by qRT-PCR,and the expression of circUSP39 was significantly elevated.Then,CCK8 assay,enzyme linked immunosorbent assay and flow cytometry were performed to evaluate the effects of circUSP39 dysfunction on cardiomyocyte proliferation,oxidative stress,inflammatory response and apoptosis induced by H/R.2.Studying the molecular mechanism of circUSP39 regulating H/R myocardial cell injury.An online bioinformatics database was first used to analyze miRNAs with binding sites with circUSP39.Using Ago2 RIP,RNA pull down experiments and dual luciferase reporter experiments validated.Analysis of downstream genes with targeted binding sites with miRNA by online bioinformatics database star Base.In addition,overexpression vector was constructed in myocardial cell lines to explore the mechanism of action through cell function experiments.3.Clinical validation of the above findings was performed.Patients with acute ST-elevation myocardial infarction(STEMI)performing emergency percutaneous coronary intervention were in the experimental group,while non-interventional patients with coronary angiography examination were in the control group.10patients in each group were enrolled to test the levels of circUSP39,downstream target genes,oxidative stress and inflammatory indicators in the two groups.Results:1.CircUSP39 was highly expressed in blood samples of AMI patients and H/R induced cardiomyocytes.Compared with the control group,after H/R treatment,the proliferation capacity of cardiomyocytes was decreased,cellular oxidative stress and inflammatory response levels were increased,and cell apoptosis rate was increased.CircUSP39 knock down groups,the proliferation ability of cardiomyocytes was enhanced,the level of oxidative stress and inflammation was decreased,and the apoptosis rate was decreased.2.Ago2 RIP,RNA pull down assay and dual luciferase reporter assay confirmed that circUSP39 directly targeted miR-362-3p.The results of cell function rescue experiment showed that after the inhibition of miR-362-3p,the inhibitory effects of circUSP39 knockdown on oxidative stress,inflammation and apoptosis of cardiomyocytes induced by H/R were weakened,and the cell proliferation ability was decreased.In addition,star Base predicted that miR-362-3p had complementary binding sites with the 3’UTR of TRAF3.The results of qRT-PCR and western blot showed that the mRNA and protein levels of TRAF3 were highly expressed in H/R-induced cardiomyocytes.Dual luciferase reporter assay confirmed that miR-362-3p could bind to the 3’UTR of TRAF3.qRT-PCR and western blot showed that overexpression of miR-362-3p could inhibit the expression of TRAF3 mRNA and protein in cardiomyocytes AC16.However,inhibition of miR-362-3p can up-regulate the expression of TRAF3 mRNA and protein in cells.The results of cell function experiment showed that overexpression of miR-362-3p could enhance the proliferation of cardiomyocytes induced by H/R,and inhibit the oxidative stress,inflammation and apoptosis of cells induced by H/R.Overexpression of TRAF3 at the same time reversed this effect.Furthermore,qRT-PCR and western blot experiments detected that circUSP39 could positively regulate TRAF3 gene expression in cardiomyocytes AC16 by targeting miR-362-3p.3.In the clinical trial section,compared with the control group,circUSP39,TRAF3,oxidative stress and inflammation indicators were significantly increased,which showed statistical differences;miR-362-3p decreased significantly with statistical difference.Conclusion:1.H/R induced decreased proliferation capacity and increased apoptosis of cardiomyocytes,and stimulated oxidative stress and inflammation of cells,circUSP39promoted the above reactions.Knockdown of circUSP39 can enhance H/R-mediated proliferation of cardiomyocytes,reduce oxidative stress and inflammatory response,and inhibit cell apoptosis.2.CircUSP39 can directly target miR-362-3p,and TRAF3 is a target gene of miR-362-3p.CircUSP39 regulates H/R damage in cardiomyocyte by targeting miR-362-3p/TRAF3 axis.The circUSP39/miR-362-3p/TRAF3 axis may be a novel mechanism in the process of myocardial cell H/R injury,and this result may provide a potential therapeutic target for improving the prognosis of AMI.3.In this study,clinical experiments confirmed that oxidative stress and inflammatory factors are involved in H/R damage,and the expression of circUSP39/miR-362-3p/TRAF 3 axis in AMI patients is involved in the H/R damage process,which is consistent with the results observed in cell experiments.更多还原