【作者】 王磊；

【导师】 黄鹤；

【作者基本信息】 武汉大学 ， 内科学， 2014， 博士

【摘要】 背景：谷胱甘肽过氧化物酶1(Glutathione peroxidase1, Gpx1)是细胞内关键性的抗氧化酶,可以解毒H202为H20,对维持细胞内氧化/还原平衡发挥了关键性的作用。近来研究表明Gpx1表达失调与许多心血管疾病的发生、发展密切相关。尽管目前,许多研究者对核转录因子κB(NFκB)和活化剂蛋白-1(AP-1)对GPxl在转录水平的调控进行了深入研究,但是目前在氧化应激下Gpxl如何在转录后水平受调控的机制尚未完全阐明。一种参与了调控心脏许多生理和病理过程的转录后调控机制便是microRNA (miRNA)。近来人们对miRNA这一内源性调控因子的认识逐渐加深。作为非编码]RNA(non-coding RNA)家族新成员之一的miRNA是一类长度约为22个核苷酸的调控性小RNA分子,它可以通过]mRNA的剪切和抑制蛋白质翻译的方式负调控靶基因。区别于其他调节因素“有”或“无”的调节特点,miRNA主要表现为在数量和程度上影响其靶基因水平,且只需通过几个碱基互补便可发挥作用。miR-181家族有四个成员(miR-181a、miR-181b、miR-181c以及]miR-181d),目前研究发现]miR-181a参与了免疫、代谢、炎症、凋亡等多种生理和病理过程。氧化应激应答的miRNA是否可以通过调控抗氧化酶的表达淬灭ROS而维持细胞内氧化还原稳态或者miRNA通过调控ROS下游的某些基因参与了氧化应激相关疾病的发生是本课题关注的重点。目的：1.探讨合适浓度H2O2介导的H9c2细胞氧化应激模型,并筛选可调控GPx1表达的niRNAs分子。2.寻求合适的转染浓度；探讨来源于大鼠的miR-181a是否可调控H9c2细胞内Gpx1的表达。3.探讨]miR-181a是否通过调节Gpx1的表达参与了氧应激诱导的H9c2细胞凋亡损伤过程。方法：1.复苏培养H9c2细胞,用不同浓度H2O2(50、100、200、400、800nmol/L)孵育H9c2细胞2小时后,CCK8法评价细胞存活率,TUNEL法检测H9c2细胞凋亡；Western blot方法检测不同H202浓度诱导下Gpx1表达变化。生物信息学软件筛选出可调控Gpx1表达的miRN As,在H9c2细胞中通过荧光定量PCR验证筛选出的miRNAs,并观察不同浓度H202对miRNA表达的影响。2.用不同浓度含Cy3标记的miR模拟剂(miR mimic)转染H9c2细胞,荧光显微镜下计数阳性细胞。构建含Gpx13’UTR双报告载体和突变载体,并同niR-181a模拟剂共转染HEK293细胞,荧光照度计测定荧光值。荧光定量PCR检测转染后miR-181a的表达;Western blot方法检测转染后Gpx1的表达。3.使用筛选出的miRNA化学合成模拟物及抑制物瞬时转染H9c2细胞,在检测前2小时加入400μmol/L H2O2,实验分6组,分别为：正常对照组；H202组；H202加niR-181a模拟物组；H202加模拟物对照(miR-CTL)组,H202加miR-181a抑制剂(anti-miR-181a)组；H202加miR-181a抑制剂对照组(anti-CTL)组。通过试剂盒检测乳酸脱氢酶(LDH)及丙二醛(MDA)水平；通过流式细胞仪检测细胞的凋亡水平；DCFH-DA法观察细胞内ROS的变化水平,JC-1试剂盒检测细胞线粒体膜电位的变化。Western blot分析凋亡蛋白Bcl-2、Bax及cleaved caspase-3的表达量的变化。细胞免疫荧光观察细胞色素c的释放。结果：1.随着H202浓度的增加,H9c2细胞存活率呈现浓度依赖性下升,而凋亡率呈现浓度依赖性上升,与对照组相比,差异有统计学意义(P<0.05)；Gpxl的表达随着H202浓度的增加呈现一个先上升后下降的趋势(P<0.05)。联合多个预测软件,预测出miR-7a, miR-125a, miR-181a以及miR-423可能调控3px1的表达。荧光定量PCR检测到400μmol/LH202处理H9c2细胞2h后候选miRNAs中仅niR-181a表达较正常组细胞上升,而其他候选niRNAs下降(P<0.05)；不同浓度H202处理H9c2后,400μmol/L H2O2浓度时miR-181a的表达达到顶峰(P<0.05)。2.随着Cy3标记的miR mimic浓度的增加,其转染效率逐渐增加,50nmol/L miR mimic组的转染效率达(96.9±4.23%)。成功构建双荧光酶素报告载体以及突变体,实验组的报告荧光比对照组明显下降(*P<0.01),而突变体的荧光强度下降不明显,差异无统计学意义。进一步研究发现转染：miR-181a抑制剂后导致H9c2细胞中miR-181a的表达下调,而Gpxl的蛋白表达略有上升；相反转染miR-181a模拟剂后导致niR-181a的表达上调,而Gpx1的蛋白表达明显下调。3.与相应对照组相比,miR-181a抑制剂组H9c2细胞的LDH、MDA、细胞的凋亡率、细胞内活性氧(ROS)的水平、Bax、cleaved caspase-3的表达量及线粒体向细胞质释放的细胞色素c均明显下降,细胞内线粒体膜电位水平及Bcl-2的表达量升高,而miR-181a模拟剂却拮抗了上述变化(P<0.05)。结论：1.400μmol/LH2O2浓度为最佳氧化应激模型浓度；miR-181a和Gpx1对H202的刺激呈现相反趋势。结合生物信息学,推测niR-181a可调控Gpx1的表达。2.50unol/L miR mimic为合适转染浓度。miR-181a在氧应激介导的心肌细胞凋亡中扮演了关键性的作用,并可负性调控Gpx1的表达。3.niR-181a能够增加H2O2诱导的氧化应激条件下H9c2心肌细胞损伤,促进H9c2,心肌细胞凋亡。]miR-181a通过调控Gpx1的表达在H202介导的H9c2细胞线粒体凋亡途径中扮演了关键性的作用。更多还原

【Abstract】 Background:Glutathione peroxidase-1(GPx1) is a pivotal intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. Recent findings indicate that the lack of GPxl contributes to the risk of atherosclerosis and cardiovascular disease. The transcriptional regulation of GPx1expression in oxidative stress via nuclear factor κB (NFκB) and activator protein1(AP-1) has been shown by many investigators. However, the post-transcriptional mechanism of Gpxl in response to H2O2-mediated oxidative stress in cardiomyocytes has not been thoroughly studied. MicroRNAs (miRNAs) are endogenous regulators of gene expression.It was reported that binding sites of target mRNAs with as few as7bp of complimentarity (seed sequence) to the miR5’end are sufficient to confer regulation in animal.The miR-181family has four members(miR-181a, miR-181b, miR-181c and miR-181d) whose seed region is complementary to a few hundred identified mRNA to date, enabling miR-181a to regulate immune response, metabolic pathways, inflammation responses, apoptosis. miRNAs can be used as powerful tools to modulate a functional phenotype that involves the participation of multiple proteins as in the case of ROS-mediated events.This raises a question whether the oxidative stress-responsive miRNAs have a role in altering the antioxidant gene expression that quench ROS to maintain redox homeostasis or control the genes downstream of H2O2stimulation that mediate the effects of oxidative stress.Objective:1. To explore appropriate concentrations of H2O2to assess the H2O2-mediated oxidative stress on H9c2cells and select several predicted miRNAs could regulate Gpxl expression in H9c2cells.2. To experimentally validate miR-181a can target Gpxl mRNA in H9c2cells.3. To demonstrate that the expression of miR-181a participated in oxidative stress-induced H9c2cells apoptosis by targeting Gpxl.Methods:1. The cultured H9c2cells were treated with different concentration (50,100,200,400,800μmol/L) of H2O2for2h.Then, CCK8assay was used to determine cell viability and the extent of apoptosis was detected by TUNEL assays. Exogenous H2O2induced endogenous Gpxl expression change in H9c2cells was assesed by Western blot. Validation of predicted miR-181a by current bio informatics were performed by real-time PCR and the expression changes of miRNAs in different concentrations of H2O2was also determined by real-time PCR.2. H9c2cells were transfected with different concentration of Cy3labeled miR mimic and the positive cells were counted using a fluorescent microscope. pmiR-RB-REPORTTM luciferase construct with the Gpxl3’-UTR and mutant version of pmiR-RB-Gpxl-3’-UTR-mut with3bp mutation within the seed region were generated.Then, they were co-transfected with miR-181a mimics to HEK293cells,respectively. After48h post-transfection, cells were lysed and assayed for luciferase activities using the Dual Glo Luciferase Assay System After transduced with miR-181a mimics and inhibitor,the expression changes of miR-181a and Gpxl were determined by real-time PCR and Western blot, respectively.3. H9c2cells were transfected with miR-181a mimics or miR-181a inhibitor, H2O2(400μmol/L) was added for the last2h. H9c2cells were divided into the following6groups:control(untransfected cells), H2O2,miR-control(miR-CTL), miR-181a, miR-181a inhibitor control (anti-CTL), and miR-181a inhibitor(anti-miR-181a) groups. The changes of apoptosis in H9c2cells were quantitatively assayed with Annexin V and PI double staining by Flow cytometric. LDH and MDA were simultaneously measured. The changes in intracellular ROS was detected by DCFH-DA staining. The measurement of mitochondrial membrane Potential was detected by using JC-1probe.The expression of Bcl-2, Bax, cleaved caspase-3in H9c2cells were detected by Western blotting. Immunofluorescent detection of intracellular cytochrome c localization was used by fluorescent microscopy.Results:1. High concentrations (100-800μmol/L) increased H9c2cell death in a dose-dependent manner after2h treatment and increasing H2O2concentrations also induced an increase in TUNEL-positive apoptotic in a dose-dependent manner(P<0.05). A significant increase in protein expression was observed in cells treated with H2O2(100μmol/L and200μmol/L), whereas400uM H2O2damage the antioxidant enzymes system resulting in Gpxl expression decreased(P<0.05). The intersection of three algorithms identified four potential miRNAs candidates:miR-7a,125a,181a and423. At the apoptotic concentration of H2O2, only miR-181a expressions were increased compared with normal group, whereas other’s expressions decreased (P<0.05). increase in miR-181a expression came to a peak at approximately400μmol/L (P<0.05).2. As increasing concentration of Cy3labeled miR mimic,the transfection efficiency increased. The transfection efficiency in50nM group was96.9±4.23%. A significant decrease in relative luciferase activity was observed when pmiR-RB-Gpxl-3’-UTR was co-transfected with miR-181a mimics as compared with the scrambled miRNAs, and the miR-181a mimics-mediated suppression was abolished by the mutation of the3’-UTR miR-181a binding site. The mimics of miR-181a resulted in decrease of Gpx1protein expression, whereas the miR-181a inhibitor protected the miR-181a mimics-mediated inhibition of Gpxl expression.3. Compared with anti-CTL group, the release of LDH and the content of MDA, the production of ROS, the apoptosis rates,Bax,cleaved caspase-3expression and the release of cytochrome c from the mitochondrial intermembrane space into the cytoplasm were significantly decreased in anti-miR-181a group (P<0.05) and the expression of Bcl-2and mitochondrial membrane potential was correspond increased in anti-miR-181a group while upregulation of miR-181a exacerbated it(P<0.05).Conclusion:1.400∴mol/L concentration of H2O2was used to assess the H2O2-mediated effect on H9c2cells. H2O2increased miR-181a expression in H9c2cells, whereas,Gpxl expression was downregulated after H2O2stimulation. With the help of current bioinformatics, we predicted miR-181a may target Gpxl.2.50nM was a proper concentration for miR mimic transfection. miR-181a plays a key role in oxidative stress-induced cardiomyocyte apoptosis and miR-181a was a negative regulator of Gpxl.3. That inhibition of miR-181a confers cardiac protection against oxidative stress-induced H9c2cell apoptosis through the direct target Gpxl expression and reduces ROS generation, which are important for the maintenance of mitochondrial membrane integrity and the inhibition of mitochondrial apoptotic pathway under oxidative stress conditions.更多还原