幽门螺杆菌感染相关microRNAs的筛选、鉴定及功能研究
|
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。
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.
Methods
1. Screening and identification of H. pylori-induced miRNAs
The 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 infection
miRNAs 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 infection
The 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 .
Results
1. Screening and identification of H. pylori-induced miRNAs
After 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 infection
2.1 Signal pathway for miR-155 up-regulation in H. pylori infection
The 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 targets
IKK-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 infection
miR-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 infection
3.1 Signal pathway for miR-146a up-regulation in H. pylori infection
Multi-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 targets
IRAK1、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 infection
miR-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.
Conclusions
1. 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.
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。
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.
Methods
1. Screening and identification of H. pylori-induced miRNAs
The 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 infection
miRNAs 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 infection
The 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 .
Results
1. Screening and identification of H. pylori-induced miRNAs
After 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 infection
2.1 Signal pathway for miR-155 up-regulation in H. pylori infection
The 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 targets
IKK-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 infection
miR-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 infection
3.1 Signal pathway for miR-146a up-regulation in H. pylori infection
Multi-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 targets
IRAK1、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 infection
miR-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.
Conclusions
1. 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.
引文
[1] Wang KX, Chen L. Helicobacter pylori L-form and patients with chronic gastritis[J]. World J Gastroenterol, 2004;10(9):1306-1309.
[2] Nagata J, kijima H, Takagi, et al. Helicobacter pylori induced chronic active gastritis in p53-knockout mice[J]. Int J Mol Med, 2004;13(6):773-777.
[3] Ohata H, Kitauchi S, Yonechi M, et al. Progression of chronic atrophic gastritis associated with Helicobacter pylori infection increases risk of gastric cancer[J]. Int J Cancer, 2004;109(1):138-143.
[4] Uemuxa N, Okamoto S, Yamamoto S, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med, 2001;345(11):784.
[5] Liew, F. Y., D. Xu, E. K. Brint, et al. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev. Immunol. 2005;5: 446–458.
[6] Han, J., Y. Lee, K. H. Yeom, et al. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex[J]. Cell, 2006;125: 887–901.
[7] Ambros V. The functions of animal microRNAs. Nature 2004;431:350-5
[8] Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834-8
[9] Pfeffer S, Zavolan M, Gr?sser FA, et al. Identification of virus-encoded microRNAs. Science, 2004;304(5671): 734-736.
[10] Cui C, Griffiths A, Li G, et al. Prediction and Identification of Herpes Simplex Virus 1-Encoded MicroRNAs[J]. J Virol, 2006;80(11): 5499-5508.
[11] Sullivan CS, Ganem D. MicroRNAs and viral infection. Mol Cell, 2005;20(1): 3-7.
[12] Lecellier CH, Dunoyer P, Arar K, et al. A Cellular MicroRNA Mediates Antiviral Defense in Human Cells. Science, 2005;308(5721): 557-560.
[13] Chen X M, Splinter P L, O Hara S P, et al. A cellular micro-RNA,let-7i, regulates Toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection[J]. J Biol Chem, 2007;282(39): 28929～28938.
[14] Odenbreit S, Puls J, Sedimaier B, et al. Translocation of Helicobacter pylori CagA protein in gastric epithelial cells by type IV secretion[J]. Science, 2000;287:1497-150.
[15]柯杨,宁涛,高燕等,人胃粘膜上皮细胞原代培养及其生物学鉴定[J],解剖学报,1992;23(4):384-389
[16]柯杨,宁涛,王冰等,人胃粘膜上皮细胞系GES-1的建立及其生物学特性[J],中华肿瘤杂志,1994;16(1):7-10
[17] XG Fan, A Chua, SJ Fan, et al. Increased gastric production of interleukin-8 and tumour necrosis factor in patients with helicobacter pylori intection[J]. J Clin Patho li.1995;48: 133
[18] Uefuji K, Ichikura T, Schinomiya N, et al. Induction of apoptosis by JTE2522, a specific cyclooxygenase-2 inhibitor, in human gastric cancer cell lines[J]. Anticancer Res, 2000;20 (5) :4279 - 4284.
[19] Guo XL, Wang LE, Du SY, et al. Association of cyclooxygenase-2 expression with Hp cagA infection in gastric cance[J]. World J Gastroenterol, 2003;9 (4) : 246 - 249.
[20] Sung JJ, LeungWK, GoMY, et al. Cyclooxygenase-2 expression in Hp associated premalignant and malignant gastric lesions[J]. Am J Pathol, 2000;157 (3) : 729 - 735.
[21] Hu PJ, Yu J, Zeng ZR, et al. Chemoprevention of gastric cancer by celecoxib rats[J]. Gut, 2004, 53 (2) : 195-200.
[22] Samaka RM, Abdou AG, Abd Eiwahed MM, et al. Cyclooxygenase-2 expression in chronic gastritis and gastric carcinoma, correlation with prognostic parameters[J]. J Egypt N atl Cancer Inst, 2006, 18 (4) : 363-374.
[23] Islam N, Haqq I TM, Jepsen KJ, et al. Hydrostatic pressure induces apop tosis in human chondrocytes from osteoarthritic cartilage through up-regulation of tumor necrosis factor-alpha,inducible nitric oxide synthase, p53, c-myc, and bax-alpha, and suppression of bcl-2[J]. J Cell B iochem , 2002, 87 ( 3 ) : 266-278.
[24] Jorge O, Cuello Carr IóN FD, Jorge A, et al. Helicobacter pylori infection affects the expression of PCNA, p53, c-erbB-2 and Bcl-2 in the human gastric mucosa[J]. Rev Esp Enferm Dig, 2003, 95 (2) : 972104, 89296.
[25] Fossl IEN E. Molecular pathology of cyclooxygenase-2 in neoplasia[J]. Ann Clin Lab Sci, 2000, 30 (1) : 3221.
[26] Crabtree J E, Courtm, Aboshkiwa MA, et al. Gastric mucosal cytokine and epithelial cell responses to Helicobacter pylori infection in Mongolian gerbils[J]. J Pathol, 2004, 202 (2) : 197-207.
[27] Moss SF, Sord Illo EM, Abdalla AM, et al. Increased gastric epithelial cell apoptosis associated with colonization with cagA+ Helicobacter pylori strains[J]. Cancer Res, 2001, 61 ( 4 ) :1406-1411.
[28] Tang H, Wang J, Ba I F, et al. Positive correlation of osteopontin, cyclooxygenase-2 and vascular endothelial growth factor in gastric cancer[J]. Cancer Invest, 2008 , 26 (1) : 60-67.
[29] Ol Iveira MJ, Costa AC, Costa AM, et al. Helicobacter pylori induces gastric ep ithelial cell invasion in a c2Met and typeⅣsecretion system-dependent manner[J]. J B iol Chem , 2006, 281(46) : 34888-34896.
[30]沈洪,孙为豪,吴爱娟等,幽门螺杆菌对大鼠胃上皮细胞环氧合酶- 2表达的影响[J],中国病理生理杂志,2007, 23 (3) : 595– 597
[31]李琦,范忠泽,孙珏等,幽门螺杆菌诱导人胃癌MKN45细胞COX-2表达的信号转导研究[J],肿瘤,2009,29(2):108-112
[32] RomanoM, RicciV, MemoliA, et al. Helicobacter pylori up - regulates cyclooxygenase - 2 mRNA exp ression and prostaglandin E2 synthesis in MKN 28 gastric mucosal cells in vitro[J]. J Biol Chem, 1998, 273 ( 44 ) : 28560-28563.
[33] Chia-Hsin Chan, Chia-Cheng Ko, Jan-Gowth Chang, et al. Subcellular and Functional Proteomic Analysis of the Cellular Responses Induced by Helicobacter pylori[J]. Molecular & Cellular Proteomics, 2006;5:702–713.
[34] Zhi-Fang Liu, Chun-Yan Chen, Wei Tang, et al. Gene-expression profiles in gastric epithelial cells stimulated with spiral and coccoid Helicobacter pylori[J]. Journal of Medical Microbiology, 2006;55:1009–1015
[35] Lee RC , Feinbaum RL , Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J] . Cell , 1993 ,75 (5) :843-854.
[36] Reinhart SJ , Slack FJ , Basson M , et a1. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans [J] . Nature , 2000 ,403 (6772) :901-906.
[37] Bentwich I , Avniel A , Karov Y, et al . Identification of hundreds of conserved and nonconserved human microRNAs[J] . Nat Genet , 2005 ,37 (7) :766-770.
[38] Berezikov E , Guryev V , van de Belt J , et al . Phylogenetic shadowing and computational identification of human microRNA genes[J] . Cell , 2005 ,120 (1) : 21-24.
[39] Miranda KC , Huynh T , Tay Y, et al . A pattern-based method for the identification of microRNA binding sites and their corresponding heteroduplexes[J] . Cell , 2006 ,126 (6) : 1203-1217.
[40] O Connell R M, Taganov K D, BoldinMP, et al. MicroRNA-155 is induced during the macrophage inflammatory response[J]. Proc Natl Acad Sci USA, 2007;104(5): 1604～1609
[41] Taganov K D, Boldin M P, Chang K J, et al. NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses[J]. Proc Natl Acad Sci USA, 2006;103(33): 12481～12486
[42] Mark M. Perry, Sterghios A. Moschos, Andrew E. Williams, et al. Rapid changes in microRNA-146a expression negatively regulate the IL-1_-induced inflammatory response in human lung alveolar epithelial cells[J]. The Journal of Immunology, 2008;180: 5689–5698.
[43] Tili E, Michaille J-J, Cimino A, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-αstimulation and their possible roles in regulating the response to endotoxin shock[J]. J Immunol, 2007;179(8): 5082～5089
[44] Jing Q, Huang S, Guth S, et al. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell, 2005;120 (5):623～634
[45] D'Elios MM, Andersen LP. Helicobacter pylori inflammation, immunity, and vaccines. Helicobacter 2007;12 Suppl 1:15-9
[46] Taganov KD, Boldin MP and Baltimore D. MicroRNAs and immunity: tiny players in a big field. Immunity 2007;26:133-7
[47] Sonkoly E, Stahle M and Pivarcsi A. MicroRNAs and immunity: novel players in the regulation of normal immune function and inflammation. Semin Cancer Biol 2008;18:131-40
[48] Zhao Y, Srivastava D. A developmental view of microRNA function[J] . Trends Biochem Sci , 2007 ,32 (4) :189-197.
[49] Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis[J]. Nature, 2005, 436 (7048) : 214-220
[50] O′Donnell KA,Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulateE2F1 expression[J]. Nature, 2005, 435(7043) : 839-843
[51] Kluiver J, Poppema S, de JongD, et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas[J]. J Pathol, 2005, 207 (2) : 243-249
[52] Akao Y,Nakagawa Y,Naoe T. let-7 microRNA functions as a potential growth suppressor in human colon cancer cells[J]. Bio.Pharm Bull, 2006, 29 (5) : 903-906
[53] Jopling CL, YiM, LancasterAM, et al. Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA[J]. Science, 2005, 309 (5740) : 1577-1581
[54] Metzler M, Wilda M, Busch K, Viehmann S and Borkhardt A. High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma[J]. Genes Chromosomes Cancer 2004;39:167-9
[55] Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005;65:7065-70
[56] Lee EJ, Gusev Y, Jiang J, et al. Expression profiling identifies microRNA signature in pancreatic cancer[J]. Int J Cancer 2007;120:1046-54
[57] Kluiver J, Poppema S, de Jong D, et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas[J]. J Pathol 2005;207:243-9
[58] Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis[J]. Cancer Cell 2006;9:189-98
[59] Thai TH, Calado DP, Casola S, et al. Regulation of the germinal center response by microRNA-155[J]. Science, 2007;316 (5824):604-608.
[60] Rodriguez A, Vigorito E, Clare S, et al. Requirement of bic/microRNA-155 for normal immune function[J]. Science 2007;316:608-11
[61] Mrazek J, Kreutmayer SB, Grasser FA, Polacek N and Huttenhofer A. Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells[J]. Nucleic Acids Res 2007;35:e73
[62] Gatto G, Rossi A, Rossi D, Kroening S, Bonatti S and Mallardo M. Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB pathway[J]. Nucleic Acids Res 2008;36:6608-19
[63] Lam LT, Davis RE, Pierce J, et al. Small molecule inhibitors of IkappaB kinase areselectively toxic for subgroups of diffuse large B-cell lymphoma defined by gene expression profiling[J]. Clin Cancer Res 2005;11:28-40
[64] Yin Q, Wang X, McBride J, Fewell C and Flemington E. B-cell receptor activation induces BIC/miR-155 expression through a conserved AP-1 element[J]. J Biol Chem 2008;283:2654-62
[65] Van den Berg A, Kroesen BJ, Kooistra K, et al. High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma[J]. Genes Chromosomes Cancer 2003;37:20-8
[66] Peters RT, Liao SM and Maniatis T. IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex[J]. Mol Cell 2000;5:513-22
[67] Imtiyaz HZ, Rosenberg S, Zhang Y, et al. The Fas-associated death domain protein is required in apoptosis and TLR-induced proliferative responses in B cells[J]. J Immunol 2006;176:6852-61
[68] Stanczyk J, Pedrioli DM, Brentano F, et al. Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis[J]. Arthritis Rheum 2008;58:1001-9
[69] Dorsett Y, McBride KM, Jankovic M, et al. MicroRNA-155 suppresses activation- induced cytidine deaminase-mediated Myc-Igh translocation[J]. Immunity 2008;28:630-8
[70] Martin MM, Lee EJ, Buckenberger JA, Schmittgen TD and Elton TS. MicroRNA-155 regulates human angiotensin II type 1 receptor expression in fibroblasts[J]. J Biol Chem 2006;281:18277-84
[71] Peters RT, Liao SM, Maniatis T. IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex[J].Mol Cell 2000; 5: 513-522
[72] Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbock DT, Coyle AJ, Liao SM, Maniatis T. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway[J]. Nat. Immunol. 2003; 4:491-496
[73] Heldin C H, Miyazono K, ten Dijke P. TGF-βsignaling from cell membrane to nucleus through SMAD proteins[J], Nature, 1997,390: 465-471
[74] Sekelsky J J ,Newfeld S, Raftery LA , Chartoff E H, GelbartWM. Genetic characterization and cloning of Mothers against dpp , a gene required for decapentaplegic function in Drosophila melanogaster[J]. Genetics, 1995, 139: 1347-1358
[75] Lo R S, Chen Y G, Shi Y, Pavletch N P, Massague J. The L 3 loop: a structural motif determining specific interactions between SMAD proteins and TGF-βreceptors[J]. EMBO J , 1998,17: 996-1005
[76] Zhang Y, Feng X,We R, Derynck R. Receptor-associated Mad homologues synergize as effectors of the TGF-βresponse[J]. Nature, 1996, 383: 168-172
[77] Ceppi M, Pereira PM, Dunand-Sauthier I, et al. MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells[J]. Proc Natl Acad Sci U S A 2009;106:2735-40
[78] Backhed F, Rokbi B, Torstensson E, et al. Gastric mucosal recognition of helicobacter pylori is independent of toll-like receptor 4[J]. J Infect Dis,2003; 187:829-836
[79] Moran AP, Aspinall GO. Unique structural and biological features of helicobacter pylori lipopolysaccharides[J]. Prog Clin BiolRes, 1998;397 :37-49
[80] Lepper PM, Triantafilou M, Schumann C, et al. Lipopolysaccharides from helicobacter pylori can act as antagonist s for toll-like receptor 4[J]. Cell Microbiol, 2005; 7 :519-528
[81] Gewirtz AT, Yu Y, Krishna US, et al. Helicobacter pylori flagellin evades toll-Like receptor 5-mediated innate immunity[J]. J Infect Dis, 2004;189:1914-1920
[82] Andersen-Nissen E, Smith KD, Strobe KL, et al. Evasion of toll-like receptor 5 by flagellated bacteria[J]. Proc Natl Acad Sci U. S. A, 2005;102 : 9247-9252
[83] Lina Fassi Fehri, Manuel Koch, Elena Belogolova, et al. Helicobacter pylori Induces miR-155 in T Cells in a cAMP-Foxp3-Dependent Manner[J]. PLoS ONE, 2010;3(5) e9500
[84] Jennifer E. Cameron, Qinyan Yin, Claire Fewell, et al. Epstein-barr virus latent membrane protein 1 induces cellular microRNA miR-146a, a modulator of lymphocyte signaling pathways[J], J.Virology, 2008;82(4):1946-1958
[85] Klemens Pichler, Grit Schneider, Ralph Grassmann, et al. MicroRNA miR-146a and further oncogenesis-related cellular microRNAs are dysregulated in HTLV-1- transformed lymphocytes[J]. Retrovirology, 2008;5:100
[86] Lo A K, To K F, Lo K W, et al. Modulation of LMP1 protein expression by EBV-encoded microRNAs[J]. Proc Natl Acad Sci USA, 2007, 104(41): 16164～16169
[87] K.M. Pauley, M. Satoh, A.L. Chan, M.R. Bubb, W.H. Reeves, E.K. Chan, Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients[J], Arthritis Res. Ther. 2008;10:R101.
[88] T. Nakasa, S. Miyaki, A. Okubo, M. Hashimoto, K. Nishida, M. Ochi, H. Asahara, Expression of microRNA-146 in rheumatoid arthritis synovial tissue[J], Arthritis Rheum. 2008;58:1284-1292.
[89] Walter J. Lukiw, Yuhai Zhao, Jian Guo Cui. An NF-κB-sensitive microRNA-146a- mediated inflammatory circuit in Alzheimer’s disease and in stressed human brain cells[J].The journal of Biological Chemistry.2008,17(9)
[90] Sonkoly E, Wei T, Janson PC et al. MicroRNAs. Novel regulators involved in the pathogenesis of Psoriasis? PloS ONE 2007; 2: 610.
[91] Dai R, Phillips RA, Zhang Y, et al. Suppression of LPS-induced Interferon-gamma and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism of immune modulation[J]. Blood, 2008;112(12): 4591-4597
[92] Schmelzer C, Kitano M, Rimbach G, et al. Effects of ubiquinol-10 on microRNA-146a expression in vitro and in vivo[J]. Mediators Inflamm, 2009;415437:1-7
[93] K. Yamasaki, T. Nakasa, S. Miyaki, M. Ishikawa, M. Deie, N. Adachi, Y. Yasunaga, H. Asahara, M. Ochi, Expression of MicroRNA-146a in osteoarthritis cartilage[J], Arthritis Rheum. 2009;60:1035-1041.
[94] Y. Tang, X. Luo, H. Cui, X. Ni, M. Yuan, Y. Guo, X. Huang, H. Zhou, N. de Vries, P.P. Tak, S. Chen, N. Shen, MicroRNA-146A contributes to abnormal activation of the type I interferon pathway in human lupus by targeting the key signaling proteins[J], Arthritis Rheum. 2009;60:1065-1075.
[95] F.J. Sheedy, L.A. O'Neill, Adding fuel to fire: microRNAs as a new class of mediators of inflammation, Ann. Rheum. Dis. 2008;67 Suppl 3:iii50-55.
[96] A.E. Williams, M.M. Perry, S.A. Moschos, H.M. Larner-Svensson, M.A. Lindsay, Role of miRNA-146a in the regulation of the innate immune response and cancer, Biochem. Soc. Trans. 2008;36:1211-1215.
[97] J. Hou, P. Wang, L. Lin, X. Liu, F. Ma, H. An, Z. Wang, X. Cao, MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2[J], J. Immunol. 2009;183: 2150-2158.
[1] Johnson SM, Grosshans H, Shingara J,et al. RAS is regulated by the let-7 microRNA family [J]. Cell, 2005, 120(5): 635-647
[2] Takamizawa J, Konishi H, Yanaqisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival [J]. Cancer Res, 2004, 64(11): 3753-3756
[3] Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2[J].Proc Natl Acad Sci , 2005, 102(39): 13944-13949
[4] Chan JA, Krichevsky AM, Kosik KS, et al. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells[J]. Cancer Res, 2005, 65(14): 6029-6033
[5] Fontana L, Pelosi E, Greco P, et al. MicroRNAs 17–5p-20a-106a control monocyt- opoiesis through AML1 targeting and M-CSF receptor up-regulation[J]. Nat Cell Biol,2007,9(7):775-787.
[6] Mark M. Perry, Sterghios A, Moschos, et al. Rapid Changes in MicroRNA-146a Expression Negatively Regulate the IL-1β-Induced Inflammatory Response in Human Lung Alveolar Epithelial Cells [J]. J Immunol,2008, 180(8): 5689-5698.
[7] Konstantin D, Taganov, Mark P. Boldin, et al. NF-ΚB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses [J]. Proc Natl Acad Sci, 2006,103 (33) :12481-12486.
[8] D Bhaumik, GK Scott, S Schokrpur, et al. Expression of microRNA-146 suppresses NF-ΚB activity with reduction of metastatic potential in breast cancer cells[J].Oncogene ,2008, 171: [Epub ahead of print]
[9] O’Connell RM, Taganov KD, Boldin MP, et al. MicroRNA-155 is induced during the macrophage inflammatory response[J]. Proc Natl Acad Sci, 2007,104(5):1604-1609.
[10]Fanyin Meng, Roger Henson, Hania Wehbe-Janek, et al.The MicroRNA let-7a Modulates Interleukin-6-dependent STAT-3 Survival Signaling in Malignant Human Cholangiocytes[J]. J Biol Chem, 2007, 282(11): 8256-8264.
[11]Budhu A, Forgues M, Ye QH, et al. Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment[J]. Cancer Cell ,2006,10(2):99-111.
[12]Seiki M, Nozumo Y, Bowman E, et al. Use of a cytokine gene expression signature in lung adenocarcinoma and the surrounding tissue as a prognostic classifier [J]. J Natl Cancer Inst ,2007,99(16):1257-1269.
[13]Tomimatsu S, Ichikura T, Mochizuki H. Significant correlation between expression of interleukin-1a and liver metastasis in gastric carcinoma[J]. Cancer ,2001, 91(7):1272-1276.
[14]Vincent T, Jourdan M, Sy MS, et al. Hyaluronic acid induces survival and proliferation of human myeloma cells through an interleukin-6-mediated pathway involving the phosphorylation of retinoblastoma protein[J]. J Biol Chem,2001,276(18):14728-14736.
[15]Knight B, Yeoh GC, Husk KL, et al . Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice[J]. J Exp Med, 2000,192(12):1809-1818.
[16]Qin Z, Kim HJ, Hemme J, et al. Inhibition of methylcholanthrene-induced carcinogenesis by an interferon-g receptordependent foreign body reaction[J]. J Exp Med, 2002,195:1479-1490.
[17]Enzler T, Gillessen S, Manis JP, et al. Deficiencies of GM-CSF and interferon-g link inflammation and cancer[J]. J Exp Med, 2003,197:1213-1219.
[18]Hussain SP, Trivers GE, Hofseth LJ, et al. Nitric oxide, a mediator of inflammation, suppresses tumorigenesis[J].Cancer Res 2004;64(19):6849-6853.
[19]Hussain SP, Amstad P, Raja K, et al. Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease[J].Cancer Res ,2000,60(13):3333-3337.
[20]Hussain SP, Raja K, Amstad PA, et al. Increased p53 mutation load in nontumorous human liver of Wilson disease and hemochromatosis: oxyradical overload diseases[J]. Proc Natl Acad Sci USA, 2000,97(23):12770-12775.
[21]Greten FR, Eckmann L, Greten TF, et al. IKKb links inflammation and tumorigenesis in a mouse model of colitis-associated cancer[J]. Cell ,2004,118:285-296.
[22] Karin M, Lin A. NF-κB at the crossroads of life and death [J]. Nat Immunol, 2002, 3(3): 221-227.
[23]Huang S, Robinson J B, Deguzman A, et al. Blockade of nuclear factor:κB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8[J]. Cancer Res, 2000, 60(19): 5334-5339.
[24]Maekita T, Nakazawa K, Mihara M, et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk[J]. Clin Cancer Res, 2006,12(3):989-995.
[25]Chan AO, Lam SK, Wong BC, et al. Promoter methylation of E-cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer. Gut ,2003,52(4):502-506.
[26]Issa JP, Ahuja N, Toyota M, et al. Accelerated age-related CpG island methylation in ulcerative colitis[J]. Cancer Res, 2001,61(9):3573-3577
[27]Hodge DR, Peng B, Cherry JC, et al. Interleukin 6 supports the maintenance of p53 tumor suppressor gene promoter methylation[J]. Cancer Res, 2005,65(11):4673-4682.
[28]Wehbe H, Henson R, Meng F, et al. Interleukin-6 contributes to growth in cholangiocarcinoma cells by aberrant promoter methylation and gene expression[J]. Cancer Res, 2006,66(21):10517-10524.
[29]Marnett LJ, Wright TL, Crews BC, et al. Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase[J]. J Biol Chem ,2000,275(18):13427-1343
[2] Nagata J, kijima H, Takagi, et al. Helicobacter pylori induced chronic active gastritis in p53-knockout mice[J]. Int J Mol Med, 2004;13(6):773-777.
[3] Ohata H, Kitauchi S, Yonechi M, et al. Progression of chronic atrophic gastritis associated with Helicobacter pylori infection increases risk of gastric cancer[J]. Int J Cancer, 2004;109(1):138-143.
[4] Uemuxa N, Okamoto S, Yamamoto S, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med, 2001;345(11):784.
[5] Liew, F. Y., D. Xu, E. K. Brint, et al. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev. Immunol. 2005;5: 446–458.
[6] Han, J., Y. Lee, K. H. Yeom, et al. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex[J]. Cell, 2006;125: 887–901.
[7] Ambros V. The functions of animal microRNAs. Nature 2004;431:350-5
[8] Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834-8
[9] Pfeffer S, Zavolan M, Gr?sser FA, et al. Identification of virus-encoded microRNAs. Science, 2004;304(5671): 734-736.
[10] Cui C, Griffiths A, Li G, et al. Prediction and Identification of Herpes Simplex Virus 1-Encoded MicroRNAs[J]. J Virol, 2006;80(11): 5499-5508.
[11] Sullivan CS, Ganem D. MicroRNAs and viral infection. Mol Cell, 2005;20(1): 3-7.
[12] Lecellier CH, Dunoyer P, Arar K, et al. A Cellular MicroRNA Mediates Antiviral Defense in Human Cells. Science, 2005;308(5721): 557-560.
[13] Chen X M, Splinter P L, O Hara S P, et al. A cellular micro-RNA,let-7i, regulates Toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection[J]. J Biol Chem, 2007;282(39): 28929～28938.
[14] Odenbreit S, Puls J, Sedimaier B, et al. Translocation of Helicobacter pylori CagA protein in gastric epithelial cells by type IV secretion[J]. Science, 2000;287:1497-150.
[15]柯杨,宁涛,高燕等,人胃粘膜上皮细胞原代培养及其生物学鉴定[J],解剖学报,1992;23(4):384-389
[16]柯杨,宁涛,王冰等,人胃粘膜上皮细胞系GES-1的建立及其生物学特性[J],中华肿瘤杂志,1994;16(1):7-10
[17] XG Fan, A Chua, SJ Fan, et al. Increased gastric production of interleukin-8 and tumour necrosis factor in patients with helicobacter pylori intection[J]. J Clin Patho li.1995;48: 133
[18] Uefuji K, Ichikura T, Schinomiya N, et al. Induction of apoptosis by JTE2522, a specific cyclooxygenase-2 inhibitor, in human gastric cancer cell lines[J]. Anticancer Res, 2000;20 (5) :4279 - 4284.
[19] Guo XL, Wang LE, Du SY, et al. Association of cyclooxygenase-2 expression with Hp cagA infection in gastric cance[J]. World J Gastroenterol, 2003;9 (4) : 246 - 249.
[20] Sung JJ, LeungWK, GoMY, et al. Cyclooxygenase-2 expression in Hp associated premalignant and malignant gastric lesions[J]. Am J Pathol, 2000;157 (3) : 729 - 735.
[21] Hu PJ, Yu J, Zeng ZR, et al. Chemoprevention of gastric cancer by celecoxib rats[J]. Gut, 2004, 53 (2) : 195-200.
[22] Samaka RM, Abdou AG, Abd Eiwahed MM, et al. Cyclooxygenase-2 expression in chronic gastritis and gastric carcinoma, correlation with prognostic parameters[J]. J Egypt N atl Cancer Inst, 2006, 18 (4) : 363-374.
[23] Islam N, Haqq I TM, Jepsen KJ, et al. Hydrostatic pressure induces apop tosis in human chondrocytes from osteoarthritic cartilage through up-regulation of tumor necrosis factor-alpha,inducible nitric oxide synthase, p53, c-myc, and bax-alpha, and suppression of bcl-2[J]. J Cell B iochem , 2002, 87 ( 3 ) : 266-278.
[24] Jorge O, Cuello Carr IóN FD, Jorge A, et al. Helicobacter pylori infection affects the expression of PCNA, p53, c-erbB-2 and Bcl-2 in the human gastric mucosa[J]. Rev Esp Enferm Dig, 2003, 95 (2) : 972104, 89296.
[25] Fossl IEN E. Molecular pathology of cyclooxygenase-2 in neoplasia[J]. Ann Clin Lab Sci, 2000, 30 (1) : 3221.
[26] Crabtree J E, Courtm, Aboshkiwa MA, et al. Gastric mucosal cytokine and epithelial cell responses to Helicobacter pylori infection in Mongolian gerbils[J]. J Pathol, 2004, 202 (2) : 197-207.
[27] Moss SF, Sord Illo EM, Abdalla AM, et al. Increased gastric epithelial cell apoptosis associated with colonization with cagA+ Helicobacter pylori strains[J]. Cancer Res, 2001, 61 ( 4 ) :1406-1411.
[28] Tang H, Wang J, Ba I F, et al. Positive correlation of osteopontin, cyclooxygenase-2 and vascular endothelial growth factor in gastric cancer[J]. Cancer Invest, 2008 , 26 (1) : 60-67.
[29] Ol Iveira MJ, Costa AC, Costa AM, et al. Helicobacter pylori induces gastric ep ithelial cell invasion in a c2Met and typeⅣsecretion system-dependent manner[J]. J B iol Chem , 2006, 281(46) : 34888-34896.
[30]沈洪,孙为豪,吴爱娟等,幽门螺杆菌对大鼠胃上皮细胞环氧合酶- 2表达的影响[J],中国病理生理杂志,2007, 23 (3) : 595– 597
[31]李琦,范忠泽,孙珏等,幽门螺杆菌诱导人胃癌MKN45细胞COX-2表达的信号转导研究[J],肿瘤,2009,29(2):108-112
[32] RomanoM, RicciV, MemoliA, et al. Helicobacter pylori up - regulates cyclooxygenase - 2 mRNA exp ression and prostaglandin E2 synthesis in MKN 28 gastric mucosal cells in vitro[J]. J Biol Chem, 1998, 273 ( 44 ) : 28560-28563.
[33] Chia-Hsin Chan, Chia-Cheng Ko, Jan-Gowth Chang, et al. Subcellular and Functional Proteomic Analysis of the Cellular Responses Induced by Helicobacter pylori[J]. Molecular & Cellular Proteomics, 2006;5:702–713.
[34] Zhi-Fang Liu, Chun-Yan Chen, Wei Tang, et al. Gene-expression profiles in gastric epithelial cells stimulated with spiral and coccoid Helicobacter pylori[J]. Journal of Medical Microbiology, 2006;55:1009–1015
[35] Lee RC , Feinbaum RL , Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J] . Cell , 1993 ,75 (5) :843-854.
[36] Reinhart SJ , Slack FJ , Basson M , et a1. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans [J] . Nature , 2000 ,403 (6772) :901-906.
[37] Bentwich I , Avniel A , Karov Y, et al . Identification of hundreds of conserved and nonconserved human microRNAs[J] . Nat Genet , 2005 ,37 (7) :766-770.
[38] Berezikov E , Guryev V , van de Belt J , et al . Phylogenetic shadowing and computational identification of human microRNA genes[J] . Cell , 2005 ,120 (1) : 21-24.
[39] Miranda KC , Huynh T , Tay Y, et al . A pattern-based method for the identification of microRNA binding sites and their corresponding heteroduplexes[J] . Cell , 2006 ,126 (6) : 1203-1217.
[40] O Connell R M, Taganov K D, BoldinMP, et al. MicroRNA-155 is induced during the macrophage inflammatory response[J]. Proc Natl Acad Sci USA, 2007;104(5): 1604～1609
[41] Taganov K D, Boldin M P, Chang K J, et al. NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses[J]. Proc Natl Acad Sci USA, 2006;103(33): 12481～12486
[42] Mark M. Perry, Sterghios A. Moschos, Andrew E. Williams, et al. Rapid changes in microRNA-146a expression negatively regulate the IL-1_-induced inflammatory response in human lung alveolar epithelial cells[J]. The Journal of Immunology, 2008;180: 5689–5698.
[43] Tili E, Michaille J-J, Cimino A, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-αstimulation and their possible roles in regulating the response to endotoxin shock[J]. J Immunol, 2007;179(8): 5082～5089
[44] Jing Q, Huang S, Guth S, et al. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell, 2005;120 (5):623～634
[45] D'Elios MM, Andersen LP. Helicobacter pylori inflammation, immunity, and vaccines. Helicobacter 2007;12 Suppl 1:15-9
[46] Taganov KD, Boldin MP and Baltimore D. MicroRNAs and immunity: tiny players in a big field. Immunity 2007;26:133-7
[47] Sonkoly E, Stahle M and Pivarcsi A. MicroRNAs and immunity: novel players in the regulation of normal immune function and inflammation. Semin Cancer Biol 2008;18:131-40
[48] Zhao Y, Srivastava D. A developmental view of microRNA function[J] . Trends Biochem Sci , 2007 ,32 (4) :189-197.
[49] Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis[J]. Nature, 2005, 436 (7048) : 214-220
[50] O′Donnell KA,Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulateE2F1 expression[J]. Nature, 2005, 435(7043) : 839-843
[51] Kluiver J, Poppema S, de JongD, et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas[J]. J Pathol, 2005, 207 (2) : 243-249
[52] Akao Y,Nakagawa Y,Naoe T. let-7 microRNA functions as a potential growth suppressor in human colon cancer cells[J]. Bio.Pharm Bull, 2006, 29 (5) : 903-906
[53] Jopling CL, YiM, LancasterAM, et al. Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA[J]. Science, 2005, 309 (5740) : 1577-1581
[54] Metzler M, Wilda M, Busch K, Viehmann S and Borkhardt A. High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma[J]. Genes Chromosomes Cancer 2004;39:167-9
[55] Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005;65:7065-70
[56] Lee EJ, Gusev Y, Jiang J, et al. Expression profiling identifies microRNA signature in pancreatic cancer[J]. Int J Cancer 2007;120:1046-54
[57] Kluiver J, Poppema S, de Jong D, et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas[J]. J Pathol 2005;207:243-9
[58] Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis[J]. Cancer Cell 2006;9:189-98
[59] Thai TH, Calado DP, Casola S, et al. Regulation of the germinal center response by microRNA-155[J]. Science, 2007;316 (5824):604-608.
[60] Rodriguez A, Vigorito E, Clare S, et al. Requirement of bic/microRNA-155 for normal immune function[J]. Science 2007;316:608-11
[61] Mrazek J, Kreutmayer SB, Grasser FA, Polacek N and Huttenhofer A. Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells[J]. Nucleic Acids Res 2007;35:e73
[62] Gatto G, Rossi A, Rossi D, Kroening S, Bonatti S and Mallardo M. Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB pathway[J]. Nucleic Acids Res 2008;36:6608-19
[63] Lam LT, Davis RE, Pierce J, et al. Small molecule inhibitors of IkappaB kinase areselectively toxic for subgroups of diffuse large B-cell lymphoma defined by gene expression profiling[J]. Clin Cancer Res 2005;11:28-40
[64] Yin Q, Wang X, McBride J, Fewell C and Flemington E. B-cell receptor activation induces BIC/miR-155 expression through a conserved AP-1 element[J]. J Biol Chem 2008;283:2654-62
[65] Van den Berg A, Kroesen BJ, Kooistra K, et al. High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma[J]. Genes Chromosomes Cancer 2003;37:20-8
[66] Peters RT, Liao SM and Maniatis T. IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex[J]. Mol Cell 2000;5:513-22
[67] Imtiyaz HZ, Rosenberg S, Zhang Y, et al. The Fas-associated death domain protein is required in apoptosis and TLR-induced proliferative responses in B cells[J]. J Immunol 2006;176:6852-61
[68] Stanczyk J, Pedrioli DM, Brentano F, et al. Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis[J]. Arthritis Rheum 2008;58:1001-9
[69] Dorsett Y, McBride KM, Jankovic M, et al. MicroRNA-155 suppresses activation- induced cytidine deaminase-mediated Myc-Igh translocation[J]. Immunity 2008;28:630-8
[70] Martin MM, Lee EJ, Buckenberger JA, Schmittgen TD and Elton TS. MicroRNA-155 regulates human angiotensin II type 1 receptor expression in fibroblasts[J]. J Biol Chem 2006;281:18277-84
[71] Peters RT, Liao SM, Maniatis T. IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex[J].Mol Cell 2000; 5: 513-522
[72] Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbock DT, Coyle AJ, Liao SM, Maniatis T. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway[J]. Nat. Immunol. 2003; 4:491-496
[73] Heldin C H, Miyazono K, ten Dijke P. TGF-βsignaling from cell membrane to nucleus through SMAD proteins[J], Nature, 1997,390: 465-471
[74] Sekelsky J J ,Newfeld S, Raftery LA , Chartoff E H, GelbartWM. Genetic characterization and cloning of Mothers against dpp , a gene required for decapentaplegic function in Drosophila melanogaster[J]. Genetics, 1995, 139: 1347-1358
[75] Lo R S, Chen Y G, Shi Y, Pavletch N P, Massague J. The L 3 loop: a structural motif determining specific interactions between SMAD proteins and TGF-βreceptors[J]. EMBO J , 1998,17: 996-1005
[76] Zhang Y, Feng X,We R, Derynck R. Receptor-associated Mad homologues synergize as effectors of the TGF-βresponse[J]. Nature, 1996, 383: 168-172
[77] Ceppi M, Pereira PM, Dunand-Sauthier I, et al. MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells[J]. Proc Natl Acad Sci U S A 2009;106:2735-40
[78] Backhed F, Rokbi B, Torstensson E, et al. Gastric mucosal recognition of helicobacter pylori is independent of toll-like receptor 4[J]. J Infect Dis,2003; 187:829-836
[79] Moran AP, Aspinall GO. Unique structural and biological features of helicobacter pylori lipopolysaccharides[J]. Prog Clin BiolRes, 1998;397 :37-49
[80] Lepper PM, Triantafilou M, Schumann C, et al. Lipopolysaccharides from helicobacter pylori can act as antagonist s for toll-like receptor 4[J]. Cell Microbiol, 2005; 7 :519-528
[81] Gewirtz AT, Yu Y, Krishna US, et al. Helicobacter pylori flagellin evades toll-Like receptor 5-mediated innate immunity[J]. J Infect Dis, 2004;189:1914-1920
[82] Andersen-Nissen E, Smith KD, Strobe KL, et al. Evasion of toll-like receptor 5 by flagellated bacteria[J]. Proc Natl Acad Sci U. S. A, 2005;102 : 9247-9252
[83] Lina Fassi Fehri, Manuel Koch, Elena Belogolova, et al. Helicobacter pylori Induces miR-155 in T Cells in a cAMP-Foxp3-Dependent Manner[J]. PLoS ONE, 2010;3(5) e9500
[84] Jennifer E. Cameron, Qinyan Yin, Claire Fewell, et al. Epstein-barr virus latent membrane protein 1 induces cellular microRNA miR-146a, a modulator of lymphocyte signaling pathways[J], J.Virology, 2008;82(4):1946-1958
[85] Klemens Pichler, Grit Schneider, Ralph Grassmann, et al. MicroRNA miR-146a and further oncogenesis-related cellular microRNAs are dysregulated in HTLV-1- transformed lymphocytes[J]. Retrovirology, 2008;5:100
[86] Lo A K, To K F, Lo K W, et al. Modulation of LMP1 protein expression by EBV-encoded microRNAs[J]. Proc Natl Acad Sci USA, 2007, 104(41): 16164～16169
[87] K.M. Pauley, M. Satoh, A.L. Chan, M.R. Bubb, W.H. Reeves, E.K. Chan, Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients[J], Arthritis Res. Ther. 2008;10:R101.
[88] T. Nakasa, S. Miyaki, A. Okubo, M. Hashimoto, K. Nishida, M. Ochi, H. Asahara, Expression of microRNA-146 in rheumatoid arthritis synovial tissue[J], Arthritis Rheum. 2008;58:1284-1292.
[89] Walter J. Lukiw, Yuhai Zhao, Jian Guo Cui. An NF-κB-sensitive microRNA-146a- mediated inflammatory circuit in Alzheimer’s disease and in stressed human brain cells[J].The journal of Biological Chemistry.2008,17(9)
[90] Sonkoly E, Wei T, Janson PC et al. MicroRNAs. Novel regulators involved in the pathogenesis of Psoriasis? PloS ONE 2007; 2: 610.
[91] Dai R, Phillips RA, Zhang Y, et al. Suppression of LPS-induced Interferon-gamma and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism of immune modulation[J]. Blood, 2008;112(12): 4591-4597
[92] Schmelzer C, Kitano M, Rimbach G, et al. Effects of ubiquinol-10 on microRNA-146a expression in vitro and in vivo[J]. Mediators Inflamm, 2009;415437:1-7
[93] K. Yamasaki, T. Nakasa, S. Miyaki, M. Ishikawa, M. Deie, N. Adachi, Y. Yasunaga, H. Asahara, M. Ochi, Expression of MicroRNA-146a in osteoarthritis cartilage[J], Arthritis Rheum. 2009;60:1035-1041.
[94] Y. Tang, X. Luo, H. Cui, X. Ni, M. Yuan, Y. Guo, X. Huang, H. Zhou, N. de Vries, P.P. Tak, S. Chen, N. Shen, MicroRNA-146A contributes to abnormal activation of the type I interferon pathway in human lupus by targeting the key signaling proteins[J], Arthritis Rheum. 2009;60:1065-1075.
[95] F.J. Sheedy, L.A. O'Neill, Adding fuel to fire: microRNAs as a new class of mediators of inflammation, Ann. Rheum. Dis. 2008;67 Suppl 3:iii50-55.
[96] A.E. Williams, M.M. Perry, S.A. Moschos, H.M. Larner-Svensson, M.A. Lindsay, Role of miRNA-146a in the regulation of the innate immune response and cancer, Biochem. Soc. Trans. 2008;36:1211-1215.
[97] J. Hou, P. Wang, L. Lin, X. Liu, F. Ma, H. An, Z. Wang, X. Cao, MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2[J], J. Immunol. 2009;183: 2150-2158.
[1] Johnson SM, Grosshans H, Shingara J,et al. RAS is regulated by the let-7 microRNA family [J]. Cell, 2005, 120(5): 635-647
[2] Takamizawa J, Konishi H, Yanaqisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival [J]. Cancer Res, 2004, 64(11): 3753-3756
[3] Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2[J].Proc Natl Acad Sci , 2005, 102(39): 13944-13949
[4] Chan JA, Krichevsky AM, Kosik KS, et al. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells[J]. Cancer Res, 2005, 65(14): 6029-6033
[5] Fontana L, Pelosi E, Greco P, et al. MicroRNAs 17–5p-20a-106a control monocyt- opoiesis through AML1 targeting and M-CSF receptor up-regulation[J]. Nat Cell Biol,2007,9(7):775-787.
[6] Mark M. Perry, Sterghios A, Moschos, et al. Rapid Changes in MicroRNA-146a Expression Negatively Regulate the IL-1β-Induced Inflammatory Response in Human Lung Alveolar Epithelial Cells [J]. J Immunol,2008, 180(8): 5689-5698.
[7] Konstantin D, Taganov, Mark P. Boldin, et al. NF-ΚB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses [J]. Proc Natl Acad Sci, 2006,103 (33) :12481-12486.
[8] D Bhaumik, GK Scott, S Schokrpur, et al. Expression of microRNA-146 suppresses NF-ΚB activity with reduction of metastatic potential in breast cancer cells[J].Oncogene ,2008, 171: [Epub ahead of print]
[9] O’Connell RM, Taganov KD, Boldin MP, et al. MicroRNA-155 is induced during the macrophage inflammatory response[J]. Proc Natl Acad Sci, 2007,104(5):1604-1609.
[10]Fanyin Meng, Roger Henson, Hania Wehbe-Janek, et al.The MicroRNA let-7a Modulates Interleukin-6-dependent STAT-3 Survival Signaling in Malignant Human Cholangiocytes[J]. J Biol Chem, 2007, 282(11): 8256-8264.
[11]Budhu A, Forgues M, Ye QH, et al. Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment[J]. Cancer Cell ,2006,10(2):99-111.
[12]Seiki M, Nozumo Y, Bowman E, et al. Use of a cytokine gene expression signature in lung adenocarcinoma and the surrounding tissue as a prognostic classifier [J]. J Natl Cancer Inst ,2007,99(16):1257-1269.
[13]Tomimatsu S, Ichikura T, Mochizuki H. Significant correlation between expression of interleukin-1a and liver metastasis in gastric carcinoma[J]. Cancer ,2001, 91(7):1272-1276.
[14]Vincent T, Jourdan M, Sy MS, et al. Hyaluronic acid induces survival and proliferation of human myeloma cells through an interleukin-6-mediated pathway involving the phosphorylation of retinoblastoma protein[J]. J Biol Chem,2001,276(18):14728-14736.
[15]Knight B, Yeoh GC, Husk KL, et al . Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice[J]. J Exp Med, 2000,192(12):1809-1818.
[16]Qin Z, Kim HJ, Hemme J, et al. Inhibition of methylcholanthrene-induced carcinogenesis by an interferon-g receptordependent foreign body reaction[J]. J Exp Med, 2002,195:1479-1490.
[17]Enzler T, Gillessen S, Manis JP, et al. Deficiencies of GM-CSF and interferon-g link inflammation and cancer[J]. J Exp Med, 2003,197:1213-1219.
[18]Hussain SP, Trivers GE, Hofseth LJ, et al. Nitric oxide, a mediator of inflammation, suppresses tumorigenesis[J].Cancer Res 2004;64(19):6849-6853.
[19]Hussain SP, Amstad P, Raja K, et al. Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease[J].Cancer Res ,2000,60(13):3333-3337.
[20]Hussain SP, Raja K, Amstad PA, et al. Increased p53 mutation load in nontumorous human liver of Wilson disease and hemochromatosis: oxyradical overload diseases[J]. Proc Natl Acad Sci USA, 2000,97(23):12770-12775.
[21]Greten FR, Eckmann L, Greten TF, et al. IKKb links inflammation and tumorigenesis in a mouse model of colitis-associated cancer[J]. Cell ,2004,118:285-296.
[22] Karin M, Lin A. NF-κB at the crossroads of life and death [J]. Nat Immunol, 2002, 3(3): 221-227.
[23]Huang S, Robinson J B, Deguzman A, et al. Blockade of nuclear factor:κB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8[J]. Cancer Res, 2000, 60(19): 5334-5339.
[24]Maekita T, Nakazawa K, Mihara M, et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk[J]. Clin Cancer Res, 2006,12(3):989-995.
[25]Chan AO, Lam SK, Wong BC, et al. Promoter methylation of E-cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer. Gut ,2003,52(4):502-506.
[26]Issa JP, Ahuja N, Toyota M, et al. Accelerated age-related CpG island methylation in ulcerative colitis[J]. Cancer Res, 2001,61(9):3573-3577
[27]Hodge DR, Peng B, Cherry JC, et al. Interleukin 6 supports the maintenance of p53 tumor suppressor gene promoter methylation[J]. Cancer Res, 2005,65(11):4673-4682.
[28]Wehbe H, Henson R, Meng F, et al. Interleukin-6 contributes to growth in cholangiocarcinoma cells by aberrant promoter methylation and gene expression[J]. Cancer Res, 2006,66(21):10517-10524.
[29]Marnett LJ, Wright TL, Crews BC, et al. Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase[J]. J Biol Chem ,2000,275(18):13427-1343