miR-214对心肌细胞凋亡的影响及其分子机制研究

发布时间：2018-09-16 21:50

【摘要】：背景：随着现代生活方式的改变、生活节奏的加快和人口老龄化的日益严重,心脏疾病发病率的升高和发病的普遍性、年轻化将日益突出。心肌细胞凋亡并不是某一个独立的心脏疾病,而是作为一个病理过程参与了多种心脏疾病的发生和发展,缺血性心肌病、心律失常、先天性心脏病、心脏过负荷、心肌的病毒感染、心肌重塑及心力衰竭等疾病均与其有着密切的关系。然而,目前对心肌细胞凋亡尚缺乏足够深入的理解,引起凋亡的多种因素、各因素间的相互作用以及下游信号通路的传递均有待进一步深入研究。miRNA是非编码RNA的一类,是由内源基因编码的单链RNA分子,长度约为18-24个核苷酸。目前的研究已经表明这类小分子物质在基因调控中起着广泛的作用。对于心肌细胞凋亡而言,阐明miRNA是如何通过某些途径调控这一复杂过程是非常必要的,值得进一步探索。本研究希望能研究某个miRNA在心肌细胞凋亡的调控中扮演的作用。目的：本研究采用real time PCR的方法验证miR-214在心肌细胞接受氧化应激后的表达情况。根据流式细胞技术及western Blot等实验方法表明miR-214在心肌细胞中的生物学功能。并且通过生物信息学的方法,对miR-214的下游靶点进行预测并实验验证。结合miR-214转基因小鼠模型,进一步研究miR-214调控心肌细胞凋亡的分子机制。方法：1.分别以不同浓度和不同作用时间的过氧化氢刺激大鼠原代心肌细胞,模拟体内心肌细胞接受氧自由基氧化应激的过程,采用real time PCR检测miR-214的表达变化情况； 2.在心肌细胞内过表达miR-214后再予适当浓度过氧化氢处理,与对照组相比,通过流式细胞术和western blot的方法分别检测凋亡细胞数的比例以及相关蛋白表达情况； 3.使用在线预测软件对miR-214进行靶基因预测,然后利用real time PCR、 western Blot等方法验证靶基因mRNA以及蛋白表达量是否在miR-214过表达后发生相应变化,并利用荧光素酶双报告系统对miR-214的靶序列进行验证； 4.结合miR-214转基因小鼠模型,利用real time PCR、western blot及免疫组化等方法再次从动物水平验证miR-214下游靶基因并分析其调控凋亡过程的具体分子机制。结果：1.不同浓度的过氧化氢同时作用心肌细胞6小时,随着浓度的提高,miR-214的表达量呈现逐渐下降的趋势。经统计检验,心肌细胞经过氧化氢应激时miR-214表达量明显低于正常心肌细胞。100uM终浓度的过氧化氢分别作用心肌细胞予不同的时间,选取Oh、1h、2h、4h、8h及12h为检测点,随着氧化应激作用时间的延长,miR-214的表达量呈逐渐降低的趋势。 2.体外转染miR-214mimic至心肌细胞使miR-214过表达,然后再分别予0uM(NC)和100uM浓度的过氧化氢处理诱导其凋亡。通过流式细胞技术和western blot实验表明,miR-214过表后的心肌细胞具有较低的细胞凋亡率,凋亡相关蛋白active-caspase3及cleaved PARP的表达也较对照组低。 3.应用在线靶基因预测软件,预测在大鼠原代心肌细胞内,bim、bak1、bax、 bnip3和casp7都有可能是miR-214的靶基因,进一步通过real time PCR和western Blot实验检测过表达miR-214后mRNA水平和蛋白水平均有下调的有bim、bax及casp7。接下来的荧光素酶双报告系统分析进一步表明它们有可能是miR-214的直接作用靶点,其3’-UTR中都存在与miR-214结合的作用位点。 4.通过构建miR-214转基因小鼠,利用real time PCR、western Blot及免疫组化等方法在体内验证了pten、puma在mRNA和蛋白水平的表达较野生小鼠相比均有所下调。结论：1.过氧化氢刺激心肌细胞,miR-214的表达量会随着时间的增加或浓度的增大呈现逐渐下降的趋势。 2.体外过表达miR-214mimic增强了心肌细胞的抗凋亡能力。 3.在影响凋亡的具体机制上,miR-214可能是同时通过多个下游靶点的协同作用实现的。
[Abstract]:BACKGROUND: With the change of modern life style, the acceleration of life rhythm and the increasing severity of aging population, the increasing incidence and prevalence of heart diseases will become increasingly prominent. Cardiomyocyte apoptosis is not an independent heart disease, but a pathological process involved in the occurrence of a variety of heart diseases. It is closely related to ischemic cardiomyopathy, arrhythmia, congenital heart disease, cardiac overload, myocardial viral infection, myocardial remodeling and heart failure. MicroRNAs are a class of non-coding RNAs, single-stranded RNA molecules encoded by endogenous genes and about 18-24 nucleotides in length. Recent studies have shown that these small molecules play an extensive role in gene regulation. For cardiomyocyte apoptosis, how microRNAs communicate is clarified. It is necessary to regulate this complex process through some pathways, and it is worth further exploring. We hope to study the role of a certain microRNA in the regulation of cardiomyocyte apoptosis.
AIM: To investigate the expression of microRNAs-214 in cardiomyocytes after oxidative stress by real-time PCR. The biological function of microRNAs-214 in cardiomyocytes was demonstrated by flow cytometry and Western Blot. The downstream target of microRNAs-214 was predicted and tested by bioinformatics method. Methods: 1. Rat primary myocardial cells were stimulated by hydrogen peroxide at different concentrations and at different time of action, and the process of oxygen free radical oxidative stress was simulated in vivo. The real-time PCR was used to detect microarray-214. The change of expression;
2. Mi-214 was overexpressed in cardiomyocytes and then treated with hydrogen peroxide at appropriate concentration. Compared with the control group, apoptotic cells were detected by flow cytometry and Western blot respectively.
3. Using on-line prediction software to predict the target gene of microRNA214, and then using real-time PCR, Western Blot and other methods to verify whether the target gene mRNA and protein expression changes after overexpression of microRNA214, and using luciferase double reporting system to verify the target sequence of microRNA214;
4. Combining with the transgenic mice model of microarray-214, the downstream target genes of microarray-214 were identified by real-time PCR, Western blot and immunohistochemistry, and the specific molecular mechanism of microarray-214 regulating apoptosis was analyzed.
Results: 1. Different concentrations of hydrogen peroxide at the same time on myocardial cells for 6 hours, with the increase of concentration, the expression of miR-214 showed a gradual downward trend. At the same time, the expression of Mi-214 decreased gradually with the prolongation of oxidative stress.
2. Mi-214 mimic was transfected into cardiomyocytes to overexpress Mi-214 in vitro, and then induced apoptosis by 0 uM (NC) and 100 uM hydrogen peroxide respectively. Flow cytometry and Western blot showed that the cardiomyocytes overexpressed by Mi-214 had a lower apoptosis rate, and the apoptosis-related proteins, active-caspase 3 and cleaved PARP, were found to be associated with apoptosis. The expression was also lower than that in the control group.
3. Using on-line target gene prediction software, it is predicted that bim, bak1, bax, BNIP 3 and CASP7 may be target genes of microarray-214 in rat primary myocardial cells. Further real-time PCR and Western Blot assay were used to detect the down-regulation of mRNA and protein levels of microarray-214 after overexpression of bim, Bax and casp7. The report system analysis further indicated that they may be direct targets of microRNAs-214, and there are binding sites with microRNAs-214 in their 3'-UTR.
4. The expression of PTEN and puma in the mRNA and protein levels of transgenic mice was down-regulated compared with wild mice by real-time PCR, Western Blot and immunohistochemistry.
Conclusion: 1. The expression of microarray-214 in cardiomyocytes stimulated by hydrogen peroxide decreased gradually with the increase of time or concentration.
2. overexpression of miR-214mimic enhances the anti apoptotic ability of cardiomyocytes.
3. Mi-214 may be synergistic with multiple downstream targets in the specific mechanism of apoptosis.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R542.2

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