miR-711在心肌缺血再灌注损伤中的作用机制

发布时间：2018-04-10 11:36

本文选题：miR-711 + 细胞损伤　；参考：《吉林大学》2016年博士论文

【摘要】：背景与目的:目前,学术界在心肌细胞损伤中的研究中普遍认为,细胞内的应激反应作用下,细胞通过改变它们的基因表达程序来适应变化。细胞可以有多种方式来做出反应,这取决于损伤的严重程度和损伤所持续时间。心肌细胞是一种分化能力极其有限的细胞。对于急性的心肌损伤,会表现各种形式的心肌细胞受损,而相对于慢性损伤,心肌细胞会变得肥大和出现心肌重构。缺氧和氧化损伤,缺氧损伤后产生的活性氧(reactive oxygen species,ROS)自由基是心肌细胞遇到的最常见的损伤,从而导致心脏功能衰竭。低氧诱导因子-1(hypoxic inducible factor-1,HIF-1),作为一种主要的氧化平衡调节基因在缺氧诱导损伤中发挥转录活化的作用,并且随后调节许多基因和其他基因的表达情况从而激活存活或者死亡途径。目前这种机制仍不清楚。mi RNA是一个含有18-24个核苷酸的单链RNA小分子,它是细胞内产生的内源性转录物,并含有一个发夹结构。mi RNA通过与目标基因的3'非翻译区(3'UTR)的部分碱基配对,在转录后沉默目标基因,抑制基因表达。通过沉默各种靶m RNA,mi RNA在各种细胞进程和疾病发展过程中发挥至关重要的作用,包括细胞增殖,细胞分裂,细胞分化,凋亡,和组织发育等等。大多数mi RNA被发现存在于非编码转录的内含子区域,而很少在外显子区域发现。据估计,三分之一的基因受mi RNA调控。一种mi RNA可调节许多基因的表达。反过来,mi RNA的表达又受转录因子和表观遗传调控。mi RNAs与心肌细胞损伤:mi RNAs与氧化损伤、缺氧及营养缺失的细胞反应都有关系。敲除特定的mi RNAs,可促进基因突变动物丧失应对这些损伤的应激能力。很多mi RNAs(mi R-14,23,-24,-26,-27,-34,-103,-107,-122,-181,-208,-210,and-213,等)已经被证实与不同的损伤有关。NF-κB1(P105)是NF-κB复合物的一个组成成分,NF-κB复合物控制着DNA的转录。NF-κB1存在于几乎所有的动物细胞中,并参与细胞对胁迫,细胞因子和自由基的反应应答。NF-κB家族的抗细胞凋亡功能来自于直接参与如p53等肿瘤的抑制蛋白作用,或者由于促凋亡与抑凋亡蛋白表达的失衡表达。NF-κB的抗凋亡功能涉及线粒体途径或外源受体途径。NF-κB在急性缺氧和再灌注损伤中起保护心肌作用,但当较长时间的损伤则导致细胞死亡。我们研究发现,缺氧刺激降低心肌细胞内NF-κB1的表达。血管生成素1(angiogenin1,Ang1或ANGPT1)是血管生成素家族中的一员。Ang1是酪氨酸激酶受体Tek的主要激动剂。Ang1最初被认为是胚胎血管稳定、分支形态化和后天血管生成必需的内皮特异性配体,其作用主要是通过与Tie2受体作用,诱导丝氨酸/苏氨酸激酶Akt(或蛋白激酶B)的磷酸化。最近的研究发现,Ang1也在心脏和骨骼肌细胞以及其他类型的细胞中表达。Ang1在保护心肌细胞免受缺血再灌注损伤中起着至关重要作用,通过结合整联蛋白,进而激活整合素β1介导的细胞外信号调节激酶(ERK)磷酸化,导致caspase9的失活。我们发现,缺氧刺激迅速降低Angl在心肌细胞中的表达。关于mi R-711的研究报道甚少。非常有限的mi RNA芯片数据显示mi R-711在化学诱导损伤的组织和癌细胞中表达上调。最近研究表明,mi R-711与吡格列酮介导的抗心肌纤维化有关系。我们已经发现在心肌缺血再灌注损伤条件mi R-711的表达升高。总之,这些研究揭示了mi R-711通过调控一些基因的表达影响细胞生长、细胞应激反应、甚至细胞死亡。然而,mi R-711是如何被调控以及如何调控应激细胞死亡的机制尚不清楚。在本研究中,我们将研究HIF-1和DNA甲基化如何调控mi R-711以及mi R-711在胁迫诱导的心肌细胞死亡的影响。实验方法:低氧诱导乳小鼠心肌细胞损伤模型的建立;mi R-711 mimic和inhibitor转入H9C2细胞中,通过流式细胞术测定心肌细胞凋亡的情况,使用western blot的方法验证mi R-711是通过NF-κB,Ang1及p-38MAPK信号通路发挥作用,统计分析。每组结果均以(Mean±SD)形式表达。每组结果均使用SPSS 20.0软件进行统计评估,P0.05时认为差异具有显著性。实验结果:分离乳小鼠心肌细胞,转染mi R-711 mimic和inhibitor后,发现mi R-711可以使乳小鼠心肌细胞Ang1表达降低,使用cell ROX和MMP方法检测细胞低氧损伤,mi R-711可以促进细胞低氧损伤,差异具有统计学意义(P0.05);5-AZ抑制DNA甲基化后,在抗霉素A作用下,H9c2细胞凋亡增加,差异具有统计学意义(P0.05);此外,HIF-1在H9c2细胞中诱导凋亡增加,是由于PIK2通路激活引起的,差异具有统计学意义(P0.05)。结论:1.低氧诱导乳小鼠心肌细胞损伤后可以使细胞凋亡增加。2.mi R-711可以促进乳小鼠心肌细胞发生低氧损伤,抑制mi R-711可以减轻心肌细胞损伤。3.mi R-711通过激活PLK,NF-κB及p-38 MAPK信号通路发挥作用。4.DNA甲基化在HIF诱导的低氧心肌细胞损伤中发挥作用。
[Abstract]:Background and objective: at present, the research is generally believed that in the myocardial cell injury in academic circles, the stress response in cells, cells by altering their gene expression program to adapt to change. Cells can have a variety of ways to make the response, it depends on the injury severity and duration of myocardial cell injury. Is a kind of extremely limited cell differentiation. For acute myocardial injury, will show various forms of myocardial cell damage, relative to chronic injury, myocardial cells become hypertrophy and cardiac remodeling. Hypoxia and oxidative damage, active oxygen production after hypoxia injury (reactive oxygen, species, ROS) is a free radical in myocardial cells the most commonly encountered injury, resulting in heart failure. Hypoxia inducible factor -1 (hypoxic inducible factor-1, HIF-1), as a main oxidation balance regulating gene in Play the role of hypoxia inducible transcription activation damage, and then the expression regulation of many genes and other genes to activate survival or death pathway. The mechanism is not clear.Mi RNA is a 18-24 nucleotide single stranded RNA molecules, it is endogenous transcripts produced inside the cell, and containing a hairpin the structure of.Mi RNA with the target gene 3'untranslated region (3'UTR) part of the base pairing, target gene silencing in post transcriptional suppression of gene expression. Through silencing target m RNA and MI RNA play a crucial role in various cellular processes and disease development process, including cell proliferation, cell division, cell differentiation apoptosis and tissue development, and so on. Most of the MI RNA were found in intron region in non encoding transcription, but seldom in the exon region. It is estimated that 1/3 of the genes regulated by Mi A mi RNA control. RNA can regulate the expression of many genes. In turn, the expression of MI RNA and.Mi RNAs and epigenetic regulation of myocardial cell injury and transcription factor table: Mi RNAs and oxidative damage, are related to cellular responses to hypoxia and lack of nutrition. The specific Mi knockout RNAs gene mutation can promote animal loss to deal with these damage stress ability. Many mi RNAs (MI R-14,23, -24, -26, -27, -34, -103, -107, -122, -181, -208, -210, and-213, etc.) have been confirmed with different damage.NF- kappa B1 (P105) is a component of NF- kappa B complexes the NF- K B complex controls the transcription of DNA.NF- kappa B1 exists in almost all animal cells, and in cells to stress, inhibition of protein function of anti apoptotic response of.NF- kappa B family of cytokines and free radicals from direct participation such as p53 tumor, or as a result of apoptosis With the imbalance of anti apoptotic protein expression of.NF- kappa B anti apoptotic function involved in the mitochondrial pathway or exogenous.NF- kappa B receptor pathway plays an important role in protecting myocardium of acute hypoxia and reperfusion injury, but for a long time the damage leading to cell death. We found that hypoxia decreased expression of myocardial cells in NF- kappa B1. Angiopoietin 1 (angiogenin1, Ang1 or ANGPT1) is the angiopoietin family in.Ang1 is a member of receptor tyrosine kinase Tek mainly agonist.Ang1 was initially thought to be embryonic vascular stability, branching morphogenesis and angiogenesis after endothelial specific ligands required, its main function is through Tie2 receptors. Induction of serine / threonine kinase Akt (or protein kinase B phosphorylation). Recent studies have found that Ang1 is also expressed in heart and skeletal muscle cells and other types of cells in.Ang1 in myocardial protection Cells from plays an important role in ischemia reperfusion injury, through the combination of integrin, and activation of extracellular signal regulated integrin beta 1 mediated kinase (ERK) phosphorylation, resulting in inactivation of caspase9. We found that hypoxia rapidly reduced the expression of Angl in myocardial cells. The Research Report about mi R-711 little MI. RNA chip data showed very limited upregulation of the expression of MI R-711 in chemical induced damage to tissues and cancer cells. Recent studies show that the relationship between R-711 and MI in myocardial fibrosis mediated by pioglitazone. We have found elevated expression in myocardial ischemia reperfusion injury mi R-711. In conclusion, these studies reveal mi R-711 through the regulation of gene expression effects on cell growth, cell stress, and cell death. However, MI R-711 is to be regulated and how to regulate stress cell death The mechanism is not clear. In this study, we will study HIF-1 and DNA methylation regulation of MI R-711 and MI to R-711 in the effect of stress induced myocardial cell death. Methods: to establish mouse myocardial cell injury model induced by hypoxia; MI R-711 and mimic inhibitor into H9C2 cells, through the determination of myocardial cells apoptosis by flow cytometry, MI R-711 verification method using Western blot is through NF- kappa B, analysis of Ang1 and p-38MAPK signaling pathway play a role in statistics. The results are in each group (Mean + SD) expression. Each results were statistically evaluated using SPSS 20 software, P0.05 that the difference was significant. Results: isolated from neonatal mouse cardiomyocytes transfected with MI, R-711 mimic and inhibitor MI, found that R-711 can make the milk Ang1 mouse cardiomyocytes decreased expression of ROX and MMP using cell method to detect cell hypoxia damage Mi R-711 can promote cell injury, hypoxia injury, the difference was statistically significant (P0.05); 5-AZ inhibition of DNA methylation, the antimycin A function, H9c2 cell apoptosis increased, the difference was statistically significant (P0.05); in addition, HIF-1 in H9c2 cells induced apoptosis, as induced by the activation of PIK2 signaling, the difference was statistically significant (P0.05). Conclusion: neonatal mouse cardiomyocytes injury induced by 1. after hypoxia could increased apoptosis of.2.mi R-711 can promote hypoxia injury in myocardial cells of neonatal mice, inhibition of MI R-711 can reduce heart muscle cell damage through activation of PLK.3.mi R-711, NF- B and P-38 kappa MAPK signaling pathway.4.DNA methyl to play a role in hypoxic myocardial cell injury induced by HIF.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54

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