【作者】 刘真；

【导师】 邹全明；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2010， 博士

【摘要】 幽门螺杆菌( Helicobacter pylori, H. pylori)是世界上感染率最高的细菌之一,全球约40%-60%的人感染H. pylori。H. pylori感染是胃炎、消化性溃疡的主要病因,且与胃癌发病密切相关,WHO已正式将H. pylori列为Ⅰ类致癌因子。H. pylori致病机理非常复杂,目前认为其主要致病机制为H. pylori毒素引起的胃黏膜损害及宿主免疫应答介导的胃黏膜损伤等。机体对于H. pylori感染能够产生强烈的细胞及体液免疫,但是并不能够有效地清除H. pylori,感染状态仍然持续存在,其中的免疫调控机制仍不清楚。微小RNA (microRNAs, miRNAs)是真核生物中一类长度约22个核苷酸的非编码小分子RNA,其编码基因存在于基因组的基因间隔区或内含子中,成熟miRNA由较长的可折叠形成发夹结构的前体转录物经Dicer酶或类似的内切核酸酶加工形成。miRNA通过与靶mRNA的3’-非翻译区(3’-UTR)互补或部分互补结合,使mRNA降解或介导其翻译抑制,参与基因转录后水平调控,在细胞发育、增殖、分化和肿瘤发生等生物学行为中发挥重要作用。研究表明,miRNAs的表达作为细胞接收外源或内源压力信号后的一种早期反应,参与调控机体免疫应答。但目前关于miRNAs在细菌感染与免疫中的作用还鲜有报道。H. pylori感染引起的由胃上皮细胞等介导的固有免疫应答成为抗感染的第一道防线,因此,本研究首先建立稳定的H. pylori感染人胃上皮细胞模型;利用miRNAs芯片检测H. pylori感染前后胃上皮细胞的miRNAs表达谱变化,以表达显著差异的miRNAs为研究对象,采用Northern杂交和实时定量PCR技术对其表达进行鉴定;深入研究H. pylori感染诱导miRNAs差异表达的分子机制;并通过生物信息学预测和报告载体系统鉴定其靶基因,详细研究miRNAs在H. pylori感染中调控炎症反应的作用机制。本研究以miRNAs为切入点,展开“H. pylori感染、miRNAs变化与调控炎症”的作用模式和机制研究,对进一步阐明H. pylori感染中的免疫调控机制具有重要意义,更为利用miRNAs靶向干预或治疗炎症相关分子导致的损伤及增强对H. pylori清除提供新的思路。方法:1、H. pylori感染相关miRNAs的筛选及鉴定。建立H. pylori标准株感染人胃上皮GES-1细胞模型;利用miRNAs芯片检测H. pylori感染前后胃上皮细胞的miRNAs表达谱变化,筛选出表达显著差异的miRNAs,并采用Northern杂交和实时定量PCR技术对其在多个H. pylori感染胃上皮细胞模型以及H. pylori感染患者胃黏膜组织中的表达进行鉴定。2、miRNAs在H. pylori感染中差异表达的机制研究。以表达显著差异的miRNAs为研究对象,通过多诱导因素综合分析、启动子分析、荧光素酶实验、信号通路抑制剂实验等方法,分析miRNAs在H. pylori感染中表达变化的分子机制。3、miRNAs在H. pylori感染中的作用研究。结合生物信息学预测、荧光素酶验证实验、GFP报告载体验证实验、实时定量PCR、Western blot等方法鉴定miRNAs在胃上皮细胞中的靶基因;通过体外过表达或抑制表达miRNAs、RNA干扰、免疫荧光等实验深入研究miRNAs在H. pylori感染中调控炎症反应的作用机制。结果:1、H. pylori感染相关miRNAs的筛选及鉴定。H. pylori 26695标准株与人胃上皮细胞GES-1共培养24h后,细胞形态呈明显“蜂鸟样”改变;细胞分泌大量促炎细胞因子Interleukin-8(IL-8);表达启动炎症反应的关键酶Cyclooxygenase-2(COX-2),成功建立感染模型。miRNAs芯片结果表明,H. pylori感染引起GES-1细胞一系列miRNAs的表达改变,其中表达上调2倍的有:miR-155、miR-146a、miR-16、miR-92b、miR-30b;表达下调2倍的有:miR-324、miR-181b。以表达变化最明显的miR-155和miR-146a为研究对象,通过Northern杂交和实时定量PCR技术对其表达进行验证,结果与芯片结果一致;且miR-155和miR-146a在其他多个H. pylori感染胃上皮细胞模型中表达均明显上调(P<0.01)。此外,与H. pylori阴性的正常胃黏膜组织相比,在H. pylori感染的慢性胃炎病人胃黏膜组织中,miR-155和miR-146a的表达量分别上调了4.32倍和4.29倍(P<0.01)。2、miR-155在H. pylori感染中的上调机制和作用研究。2.1 H. pylori感染诱导miR-155高表达的信号通路研究。启动子预测结果显示,miR-155基因BIC的启动子序列中含有NF-κB和AP-1的结合位点。荧光素酶实验和信号通路抑制剂实验表明,NF-κB和AP-1信号通路均参与了miR-155的诱导表达,其中AP-1在miR-155的诱导表达中起着更关键的作用。2.2 miR-155靶基因的预测与鉴定。利用生物信息学软件预测到miR-155的靶基因:IκB kinaseε(IKK-ε)、SMAD2和Fas-associated death domain protein(FADD);构建作用靶点荧光素酶报告载体和GFP报告载体,证实了miR-155能与三个靶基因的3′-UTR结合;实时定量PCR和Western blot结果表明,miR-155可通过降解IKK-ε和SMAD2 mRNA从而抑制其蛋白表达;还可直接抑制FADD蛋白翻译影响其表达。2.3 miR-155抑制H. pylori感染中炎症因子的表达。体外过表达miR-155后,能够显著减少H. pylori感染引起的炎症因子(IL-8、Growth-related oncogene-α(GRO-α))表达(P<0.05),且这种抑制作用是通过降低NF-κB活性引起的次级效应,证明miR-155参与了H. pylori感染中炎症反应的负反馈调控。3、miR-146a在H. pylori感染中的上调机制和作用研究。3.1 miR-146a在H. pylori感染中表达上调的分子机制研究。多诱导因素综合分析显示,H. pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够诱导miR-146a的表达明显上调(P<0.01),但这类诱导因素对于miR-146a的高表达为充分非必要条件。启动子预测结果显示,miR-146a基因的启动子序列中含有多个NF-κB结合位点。荧光素酶实验和信号通路抑制剂实验表明,NF-κB信号通路在miR-146a的诱导表达中起关键作用。3.2 miR-146a靶基因的预测及鉴定。利用TargetScan、Miranda、PicTar三大靶标分析软件预测到miR-146a的靶基因:Interleukin-1 receptor-associated kinase 1(IRAK1)、TNF receptor-associated factor 6 (TRAF6)和COX-2;构建作用靶点荧光素酶报告载体和GFP报告载体,证实了miR-146a能与靶基因的3′-UTR结合;实时定量PCR和Western blot结果表明,miR-146a可通过降解IRAK1、TRAF6和COX-2 mRNA从而抑制其蛋白表达。3.3 miR-146a抑制H. pylori感染中COX-2和炎症因子的表达。体外过表达miR-146a后,能够显著减少H. pylori感染引起的炎症反应关键酶COX-2和炎症因子(IL-8、Macrophage inflammatory protein-3α(MIP-3α)、GRO-α)的表达(P<0.05),且这种对炎症因子表达的抑制作用是通过抑制NF-κB核转位从而降低NF-κB活性引起的次级效应,证明miR-146a在H. pylori感染炎症反应中发挥负反馈调控作用。结论:1、建立了H. pylori标准株感染人胃上皮GES-1细胞模型,筛选到H. pylori感染相关miRNAs;验证了H. pylori感染能够引起人胃上皮细胞株和人胃黏膜组织中miR-155和miR-146a的表达上调。2、软件分析表明了miR-155和miR-146a基因的启动子序列中含有NF-κB和/或AP-1结合位点;通过信号通路相关实验,证实了H. pylori感染诱导miR-155和miR-146a的高表达受到NF-κB和/或AP-1信号通路调节。3、预测并验证了miR-155和miR-146a在胃上皮细胞中的部分靶基因,表明miR-155和miR-146a通过作用于IKK-ε、SMAD2、FADD、IRAK1、TRAF6和COX-2等在信号转导、炎症反应、肿瘤发生等过程中发挥重要作用的关键蛋白,参与负性调控H. pylori感染引起的炎症反应。4、miR-155和miR-146a作为一类新的负反馈调节因子,与其靶基因构成全新的基因调控网络,参与H. pylori感染中炎症反应的调节过程,这为进一步阐明H. pylori感染的免疫调控机制及H. pylori的致病机制研究提供新的方向。更多还原

【Abstract】 Helicobacter pylori (H. pylori) is one of the most popular bacteria, which is closely linked to the development of gastritis, peptic ulcer diseases, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. H. pylori has been classified as Type I carcinogen by the World Health Organization. The remarkable feature of the H. pylori infection is its complicated immune response. Though strong cellular and humoral immunity is induced by H. pylori infection, the immune and inflammatory responses are unable to clear the bacteria, resulting in lifelong bacterial persistence. We are still far from unveiling the exact regulatory mechanism of this complex system.MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs that are implicated in many physiological and pathological processes as post-transcriptional repressors of gene expression. Mature miRNAs can specifically bind to 3’-UTRs of target cellular mRNA in turn triggering mRNA degradation or inhibition of translation. In general, miRNAs act as key regulators in development, differentiation, homeostasis, and cancers.As the earlier reaction of the cells receiving exogeous and endogenous pressure, miRNAs are involved in modulating immune response. However, the regulatory role of miRNAs in bacteria infection and immunity is not clear. H. pylori-induced innate immune response mediated by gastric epithelium cells acts as the first line of defense against infection. Here we firstly established stable gastric epithelium cells model infected by H. pylori, and by microarray we analyzed the expression profile of cellular miRNAs during H. pylori infection. Then we chose miRNAs which expression were significantly altered for detailed investigation objects, and identified their expressions by the quantitative RT-PCR and Northern blot assays. Subsequently, we investigated the underlying mechanism leading to miRNAs differential expression by H. pylori, and identified the potential target genes of miRNAs by bioinformatics prediction and reporter vector system, and investigated the possible roles of miRNAs as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection. Our results provided insights into the regulatory networks of H. pylori-induced inflammations. Moreover, the altered miRNAs expression may identify a potential link between miRNAs and immune regulation during H. pylori infection.Methods1. Screening and identification of H. pylori-induced miRNAsThe gastric epithelium cells model infected by H. pylori were established. The expression profile of cellular miRNAs during H. pylori infection was analyzed by microarray. Then miRNAs which expression were significantly altered were screened and identified in several infection models and in H. pylori-infected gastric mucosal tissues by the quantitative RT-PCR and Northern blot.2. Study on the mechanism of miRNAs differential expression in H. pylori infectionmiRNAs which expression were significantly altered were identified as the detailed investigation objects, and their alteration mechanism of miRNAs in H. pylori infection were analyzed by multi-stimulus analysis, promoter prediction, luciferase array and signal pathway inhibitors experiment.3. Studies on the fuction of miRNAs in H. pylori infectionThe potential targets of miRNAs in gastric epithelium cells were identified by bioinformatics prediction, luciferase reporter assay, GFP reporter assay, Realtime PCR and Western blot. Examination of miRNAs function in H. pylori infection were performed by overexpression and inhibition of miRNAs, RNAi, immunofluorescence .Results1. Screening and identification of H. pylori-induced miRNAsAfter the incubation of H. pylori 26695 with the gastric epithelium cells for 24h, induction of the scattering phenotype, IL-8 Release, and COX-2 protein expression in GES-1 cells were observed. The expression of miRNAs could be significantly altered during H. pylori infection by microarray, including the up-regulation of miR-155, miR-146a, miR-16, miR-92b, miR-30b and the down-regulation of miR-324 and miR-181b. In consensus with the findings from microarray, the results of Realtime-PCR showed that miR-155 and miR-146a in several infection models and in H. pylori-infected gastric mucosal tissues were significantly increased(P<0.01). Furthermore, miR-155 and miR-146a were highly up-regulated in H.pylori-positive patients, with 4.32 and 4.29 fold change respectively as compared with the control (P<0.01).2. Studies on the up-regulation mechanism and fuction for miR-155 in H. pylori infection2.1 Signal pathway for miR-155 up-regulation in H. pylori infectionThe promoter region of miR-155 contained putative NF-κB and AP-1 binding sites. The results of promoter analysis and inhibitor experiment showed that both NF-κB and AP-1 pathways are required for the up-regulation of miR-155 in response to H. pylori, and AP-1 plays a central role in the induction of miR-155.2.2 Prediction and identification of miR-155 targetsIKK-ε, SMAD2, and FADD are potential targets of miR-155, and miR-155 might down-regulate the target protein through mRNA degradation or translation inhibition.2.3 Inhibition of proinflammatory cytokines of miR-155 in H. pylori infectionmiR-155 mimics significantly attenuated the mRNA and protein levels of IL-8 and GRO-α(P<0.05), and the effect of miR-155 in modulating the inflammation may be as a secondary effect through diminishing NF-κB activity. miR-155 may be involved in the negative feedback regulation of inflammation.3. Studies on the up-regulation mechanism and fuction for miR-146a in H. pylori infection3.1 Signal pathway for miR-146a up-regulation in H. pylori infectionMulti-stimulus analysis showed H. pylori-related proinflammatory cytokines were not necessary to miR-146a up-regulation in H. pylori infection. Moreover, the promoter region of miR-146a contained several putative NF-κB binding sites. The results of promoter analysis and inhibitor experiment showed that NF-κB pathway is required for the up-regulation of miR-146a in response to H. pylori.3.2. Prediction and identification of miR-146a targetsIRAK1、TRAF6 and COX-2 are potential targets of miR-146a, and miR-146a might down-regulate the target protein through mRNA degradation.3.3. Inhibition of COX-2 and proinflammatory cytokines of miR-146a in H. pylori infectionmiR-146a mimics significantly attenuated the mRNA and protein levels of IL-8 , MIP-3αand GRO-α(P<0.05), and the effect of miR-146a in modulating the inflammation may be as a secondary effect through diminishing NF-κB activity. miR-146a may be involved in the negative feedback regulation of inflammation.Conclusions1. We established the gastric epithelium cells model infected by H. pylori, and analyzed the expression profile of cellular miRNAs during H. pylori infection by microarray. Then we chose miR-155 and miR-146a for detailed investigation, and identified the up-regulation in several infection cell models and in H. pylori-infected gastric mucosal tissues by the quantitative RT-PCR and Northern blot. miR-155 and miR-146a expression were positive correlated.2. Bioinformatics anaylsis indicated the promoter regions of miR-155 and miR-146a contained putative NF-κB and/or AP-1 binding sites. The results of promoter analysis and inhibitor experiment showed that NF-κB and/or AP-1 pathway were required for the up-regulation of miR-155 and miR-146a in response to H. pylori.3. As the targets of miR-155 and miR-146a, IKK-ε, SMAD2, FADD, IRAK1, TRAF6 and COX-2 were identified. miR-155 and miR-146a could significantly attenuate the mRNA and protein levels of proinflammatory cytokines induced by H. pylori infection, and the effects of miR-155 and miR-146a in modulating the inflammation may be as a secondary effect through diminishing NF-κB activity.4. miR-155 and miR-146a may function as novel negative regulators. Together with its targets, miR-155 and miR-146a may be involved in the negative feedback regulation in inflammation response of H. pylori infection. Furthermore, the altered miR-155 and miR-146a expression may establish a potential link between miRNAs and pathogenesis of H. pylori related diseases.更多还原