MicroRNA-34a对心肌细胞SH2B3表达的调控作用

发布时间：2018-04-15 22:23

本文选题：心肌纤维化 + 转化生长因子-β1　；参考：《山东大学》2016年硕士论文

【摘要】：背景心肌纤维化严重影响心脏的正常功能,在外界因素刺激下,细胞外基质中成分的异常是导致纤维化的最主要因素,而延缓或阻止纤维化的发生和进展可明显改善心肌功能。随着研究的不断深入,通过调控细胞活性来影响心肌纤维化已成为治疗该类疾病的主要目标。因此,了解心肌纤维化的发生机制及调节途径显得非常重要。当心肌细胞接受外界不良刺激后,会通过一系列步骤将信号传导入心肌细胞内部,从而影响细胞的功能和活性。SH2B3属于SH2B接头蛋白家族成员之一,影响细胞内分子的定位,可影响许多由Janus激酶和受体酪氨酸激酶介导的信号通路活性。研究表明,SH283参与造血细胞的分化,并与心肌梗死、代谢综合征、多发性硬化症、系统性红斑狼疮病、类风湿性关节炎等疾病关系相当密切。在小鼠心肌梗死模型中,敲除了SH283表达后,其纤维化程度随之升高,在当前的研究成果中,已提示在心肌梗死后纤维化过程中SH2B3发挥了一定的作用,但到目前为止,关于该分子在心肌纤维化过程中的表达调控机制还不清楚。microRN A(miRNA)是一类含21-25个碱基的非编码单链核苷酸,近年来被认为是一种新的转录后调节子,可通过与靶基因的3’非翻译区(3’untranslated region,3'UTR)相互作用来调节靶基因的表达,最终影响细胞活性。该分子的作用特点是同-miRNA可对多个靶基因进行同时调控。越来越多的证据表明,miRNA在心血管疾病,特别是心肌纤维化过程中发挥重要作用。最新有关研究已表明,在心肌梗死模型中miR-34a参与了心肌细胞收缩功能的调控,抑制其表达可提高心肌的收缩功能。目前Flister等研究均已发现,IniR-34a通过靶向调控Smad4、促进分泌纤维化相关胶原,使其在心肌梗死后纤维化区域中的表达显然上调,并最终影响了心肌纤维化过程。研究目的(1)观察转化生长因子-β1对miR-34a和SH2B3表达的作用；(2)明确miR-34a对SH2B3的调控作用。方法为了证明miR-34a可能调控SH283的表达,本课题首先体外培养心肌细胞,分别在接受转化生长因子-β1和缺氧处理后,提取总蛋白和RNA后,采用Western blot和qRT-PCR法检测SH2B3在蛋白水平和mRNA水平的表达量变化；采用qRT-PCR法检测接受以上刺激后,miR-34a的表达；在心肌细胞中转染miR-34a mimics和miR-34a inhibitor后,采用Western blot和qRT-PCR法检测SH283在蛋白水平和mRNA水平的表达量变化；构建针对miR-34a作用位点的SH283荧光素酶报告基因载体,采用荧光素酶报告基因法检测miR-34a对SH283基因3’UTR区活性的影响。结果实验结果表明,在转化生长因子-β1诱导心肌细胞作用过程中,SH2B3在mRNA和蛋白水平的表达均降低；miR-34a的表达水平升高；α-SMA蛋白的表达升高。Western blot和qRT-PCR结果显示转染miR-34a模拟物后,SH283的表达降低；转染miR-34a抑制物后SH283的表达升高。荧光素酶报告检测发现,miR-34a可通过影响SH2B33'UTR活性,调控其表达。另外,在心肌细胞接受缺氧刺激后,SH283的表达量随着刺激时间的延长而降低,miR-34a的表达则升高,这种表达的相反趋势进一步证明两者的相互调控关系。结论1.发现miR-34a可作用于SH2B3的3'UTR区,从而调控其在心肌细胞中的表达。2.在TGF-β1及缺氧条件下,miR-34a与SH2B3表现出相反的表达趋势,提示miR-34a/SH2B3参与TGF-β1及缺氧对心肌细胞的作用过程。
[Abstract]:Background myocardial fibrosis seriously affect the normal function of the heart, in the external factors under the stimulation of extracellular matrix components abnormalities are the main factors leading to the occurrence and progression of fibrosis, and delay or prevent fibrosis can significantly improve myocardial function. With the continuous deepening of research, influence of myocardial fibrosis by regulating cell activity has become the main the target for treating the diseases. Therefore, understanding the mechanism of myocardial fibrosis and regulation is very important. When the myocardial cells accept adverse external stimulation, through a series of steps to internal import myocardial cell signals, which affect cell function and activity of.SH2B3 is one of the SH2B adaptor protein family members influence intracellular localization molecules that can affect many by Janus kinase and receptor tyrosine kinase mediated signal pathway activity. The results show that SH283 is involved in hematopoiesis Cell differentiation, and myocardial infarction, metabolic syndrome, multiple sclerosis, systemic lupus erythematosus disease, rheumatoid arthritis and other diseases quite close. The mouse model of myocardial infarction, knocking down SH283 expression, the degree of fibrosis increased, in the current research, has suggested that SH2B3 to play a certain role in the process of fibrosis after myocardial infarction, but so far, the molecular process of myocardial fibrosis in the expression regulation mechanism is not clear.MicroRN A (miRNA) is a kind of non encoding single nucleotide containing 21-25 bases, in recent years is considered a new post transcriptional regulator with the target gene, 3 'untranslated region (3' untranslated region, 3'UTR) interact to regulate expression of target genes and ultimately affect the activity of cells. The molecular characteristics of the same -miRNA can be carried on the multiple target genes At the same time regulation. More and more evidence that miRNA in cardiovascular disease, especially play an important role in the process of myocardial fibrosis. The latest research has indicated that, in the model of myocardial infarction in miR-34a involved in the regulation of myocardial contractile function, inhibition of its expression can improve the systolic function of heart muscle. At present Flister research have been found IniR-34a Smad4, controlled by the target to promote the secretion of collagen, fibrosis, the expression of fibrosis after myocardial infarction in the region increased obviously, and ultimately affect the myocardial fibrosis process. The purpose of the study (1) to observe the expression of transforming growth factor beta 1 on miR-34a and SH2B3; (2) regulation clear miR-34a the SH2B3 method. In order to prove that miR-34a can regulate the expression of SH283, the first issue of myocardial cells in vitro, respectively in transforming growth factor beta 1 and after hypoxia treatment, extraction of total eggs White and RNA, expression of blot and qRT-PCR was detected by Western SH2B3 on mRNA and protein level were detected by qRT-PCR; accept the stimulation, the expression of miR-34a in myocardial cells; transfection of miR-34a mimics and miR-34a inhibitor, expression of blot and qRT-PCR were detected by Western and SH283 at protein level the level of mRNA; to construct the SH283 luciferase reporter vector miR-34a loci, effect of luciferase assay was used to detect miR-34a of SH283 gene 3 'UTR activity. The experimental results show that the transforming growth factor beta 1 induced myocardial cell process, the expression of SH2B3 at mRNA and protein levels were decreased; increase the expression level of miR-34a increased; the expression of a -SMA protein of.Western blot and qRT-PCR results showed that the transfection of miR-34a mimics, decreased the expression of SH283 ; the expression of SH283 increased transfection of miR-34a inhibitor. Luciferase reporter assay showed that miR-34a could affect the activity of SH2B33'UTR and the regulation of its expression. In addition, accept the hypoxia stimulation in myocardial cells, the expression of SH283 was decreased with time of stimulation, the expression of miR-34a was increased, the expression of the opposite trend further proved both the regulation of relationship. Conclusion 1. miR-34a can be found in the SH2B3 3'UTR area, so as to regulate the expression of.2. in myocardial cells in TGF- beta 1 and under hypoxic conditions, miR-34a and SH2B3 showed the opposite trend of expression, suggesting that miR-34a/SH2B3 in TGF- beta 1 and the effect of hypoxia on myocardial cells.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R542.23

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