幽门螺杆菌CagA抑制胃上皮细胞自噬及其感染引起的miR-222/146a上调在胃相关疾病发生中的作用机制研究
|
摘要
幽门螺杆菌(Helicobacter pylori，H. pylori)是慢性胃炎和消化性溃疡的主要致病因子，与胃粘膜淋巴组织淋巴瘤和胃癌的发病密切相关，阐明致病机制对预防和控制其感染引起的相关疾病具有重要的理论指导意义。
H. pylori的感染与致病，是一个与机体相互斗争的过程。自噬在抗病原微生物感染以及其导致的炎症中发挥重要作用。自噬受到抑制，可以促进炎性因子的释放，引起炎症反应，导致疾病的发生发展。一方面，H. pylori感染胃上皮细胞可诱导自噬,通过自噬降解病原体或其分泌的毒性分子起到保护胃上皮细胞的作用；另一方面，H.pylori为了达到感染和致病目的，存在某种机制对抗自噬。本研究提出CagA负性调控幽门螺杆菌诱导的胃上皮细胞自噬在慢性胃炎发生中的作用机制： H. pylori感染可活化胃粘膜上皮细胞内肝细胞生长因子(HGF)的受体c-Met,磷酸化c-Met可与通过Ⅳ分泌系统转运至胃上皮细胞内的CagA发生相互作用，启动了其下游的PI3K/Akt信号途径的活化,通过调节自噬通路中的信号蛋白mTOR而发挥抑制自噬的作用，从而影响到自噬对胃上皮细胞保护作用，导致CagA阳性H. pylori菌株胃上皮细胞炎症反应较重，引起慢性胃炎、消化性溃疡，甚至胃癌等疾病。
miRNAs作为一类新的癌基因或抑癌基因，其表达失衡与多种肿瘤的发生密切相关。前期研究中，我们通过芯片分析H. pylori感染胃上皮细胞miRNAs的变化，发现与肿瘤发生密切相关的miR-222表达上调，而其靶基因RECK作为一种抑癌基因，可以负性调控基质金属蛋白酶(MMP)，抑制肿瘤的生长和转移，其在胃癌组织和细胞株中表达缺失，与胃癌的预后呈负相关。因此，我们推测miR-222可能是一个与H.pylori感染和胃癌发生密切相关的癌基因。H. pylori感染能够诱导miR-222的高表达，而miR-222可能通过抑制下游抑癌基因RECK的表达，调控细胞的增殖和凋亡，促进胃癌的发生和转移。
H. pylori可以在人体特殊的复杂多变的胃环境中长期定居，其慢性持续性感染在所致疾病的发生发展中发挥着重要作用。近年来，越来越多的证据显示miRNAs在多个环节参与调控机体固有免疫应答和获得性免疫反应，在宿主抗病原微生物感染时扮演重要的角色。前期研究发现H. pylori感染相关的miR-146a通过抑制靶基因IRAK1、TRAF6和COX-2的表达，降低NF-κB的活性，从而抑制H. pylori感染导致的炎症因子的释放。为深入研究在H. pylori感染诱导miR-146a表达过程中究竟什么因素起关键作用，我们观察了H. pylori感染相关炎性细胞因子以及H. pylori毒素蛋白CagA在胃上皮细胞中对miR-146a表达的影响。
【研究目的】
1.评价H. pylori感染胃上皮细胞诱导自噬发生的过程中CagA对自噬的抑制作用；观察自噬抑制对H. pylori感染引起的炎症反应的影响；探讨CagA负性调控H. pylori诱导的胃上皮细胞自噬的机制。
2.分析H. pylori感染胃上皮细胞、胃粘膜组织以及胃癌细胞株、胃癌组织中miR-222的表达；筛选并鉴定miR-222的靶基因，探讨其致癌机制。
3.观察H. pylori相关炎性因子IL-8、TNF-α以及IL-1β刺激HGC-27细胞对miR-146a表达影响；评价miR-146a对H. pylori感染诱导炎症因子释放的影响；探讨毒力因子CagA对H. pylori感染诱导miR-146a的表达影响。
【研究方法】
1.幽门螺杆菌CagA抑制胃上皮细胞自噬及其在慢性胃炎发生中的作用机制研究
Western-blot、激光共聚焦、电镜以及流式细胞技术分别检测Hp-WT或Hp-△CagA感染人胃上皮细胞自噬模型和临床胃粘膜标本自噬水平；检测体外过表达CagA对Rapamycin诱导自噬的影响；使用化学药物法抑制自噬或利用siRNA技术阻断自噬，ELISA分别检测Hp-WT和Hp-△CagA两种菌株感染诱导炎症因子分泌的水平，荧光素酶法检测NF-κB荧光素酶报告基因活性；Western-blot检测PI3K/Akt/mTOR信号途径是否参与了H. pylori感染过程中CagA对自噬的抑制作用；利用siRNA沉默c-Met表达，Western-blot、激光共聚焦显微镜、流式细胞技术检测H. pylori感染诱导的自噬情况。
2. miR-222高表达在幽门螺杆菌感染诱发胃癌中的作用及机制研究
RT-PCR检测H. pylori感染胃上皮细胞系前后以及H. pylori感染患者胃粘膜组织、胃癌细胞株以及胃癌组织中miR-222的表达；CCK-8法检测miR-222对两种胃癌细胞株增殖的影响，分析miR-222对两种胃癌细胞株克隆形成的影响；利用双荧光素酶实验、GFP荧光抑制实验、Western blot鉴定靶基因；利用RT-PCR技术检测靶基因与miR-222胃癌组织样本中的表达水平及相关性；CCK-8法和克隆形成实验分别检测沉默RECK表达是否可以模拟过表达miR-222对胃癌细胞AGS增殖和克隆形成的影响。
3. H. pylori感染HGC-27细胞miR-146a表达调节机制初步研究
利用RT-PCR技术检测IL-8、TNF-α以及IL-1β刺激HGC-27细胞后miR-146a表达情况；siRNA沉默IL-8、TNF-α和IL-1β的表达，RT-PCR技术检测H. pylori感染后miR-146a表达；荧光素酶法检测IL-8、TNF-α或IL-1β刺激后NF-κB荧光素酶报告基因活性；分别用IL-8、TNF-α或IL-1β刺激NF-κB抑制剂(BAY-117082)处理过的HGC-27细胞，RT-PCR技术检测miR-146a表达；RT-PCR和ELISA技术分别检测过表达miR-146a对H.pylori感染炎症因子mRNA和蛋白水平； CagA阳性或阴性H. pylori菌株感染HGC-27细胞，RT-PCR技术检测miR-146a表达；转染HGC-27细胞pEGFP-C1-CagA质粒，荧光显微镜和免疫印迹检测转染效率，RT-PCR技术检测内源性高表达CagA对miR-146a的表达影响。
【主要结论】
1. CagA具有抑制H. pylori诱导胃上皮细胞自噬的作用；抑制自噬可以增强Hp-△CagA感染介导的炎症程度；CagA可能通过PI3K/Akt/mTOR信号途径介导了H. pylori感染过程中对自噬的抑制作用；c-Met分子可能参与了CagA抑制H. pylori感染胃上皮细胞自噬过程。
2.无论是体外还是体内H. pylori感染均能诱导miR-222的表达上调；miR-222在胃癌细胞株和胃癌组织中均呈高表达；RECK为miR-222的新靶基因；H. pylori感染诱导的miR-222的高表达，可能通过靶向抑制RECK蛋白的表达，来发挥促进肿瘤细胞增殖的作用；胃癌组织中，miR-222与RECK转录水平表达量呈现很好的负相关。
3. H. pylori相关炎性因子刺激胃上皮细胞HGC-27诱导miR-146a表达上调，但对于miR-146a的高表达为非必要条件； NF-κB活化参与了IL-8、 TNF-α以及IL-1β诱导miR-146a高表达；过表达miR-146a可以抑制H. pylori感染相关炎症因子的释放；H.pylori感染对miR-146a的诱导不依赖于毒素蛋白CagA。
【研究意义】
1.本研究首次提出幽门螺杆菌CagA作为一种负性调控因子，通过抑制自噬引起慢性胃炎的新观点，并在体内外从现象到机制进行了探讨，为深入理解和认识H. pylori慢性感染致病机制、寻找H. pylori感染有效防治手段提供了新的视角。
2. miR-222可能参与H. pylori感染诱发胃癌的过程，可以作为胃癌诊断或治疗的靶点，在H. pylori感染与胃癌形成的过程中发挥调节作用，研究为阐明H. pylori的致癌机制提供了新线索。
3. H. pylori感染引起的炎症免疫反应调控机制十分复杂且涉及诸多环节。miRNAs在H. pylori感染的免疫调节中发挥重要作用。本文对H. pylori感染诱导miR-146a表达的调节机制予以了初步探索，miR-146a可能成为H. pylori感染诊断、治疗和预后的新靶点。
Helicobacter pylori (H. pylori) is present in the stomach of at least half of the world’spopulation, and many studies have indicated that persistent colonization of the stomach byH. pylori causes gastric diseases such as chronic gastritis, peptic ulcer disease,mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. However, themechanism by which H. pylori induce proinflammatory responses in gastric epithelial cellshas long remained a mystery.
During H. pylori infection, host and environmental factors consist of a complex war toregulate the host response. Autophagy is thought to serve as an innate defense mechanismagainst infection. Recent studies have implicated inhibition of autophagy causes fuelinflammation. In this process, H. pylori can induce autophagy in gastric epithelial cells,which play a role in the protection of gastric epithelial cells. On the other hand, thepathogens are able to subvert autophagy as astrategy for pathopoiesis. Here, we showed thatCagA may be a negative regulated factor in autophagy induced by H. pylori. We willexplore the mechanism that H. pylori infection can activate c-Met, and then phosphorylasedc-Met interact with CagA which transport through the Ⅳ secrete system, and thenPI3K/Akt/mTOR signal way was activated to inhibit autophagy. The aim is to detect forautophagy inhibited by CagA in gastric epithelium cells and the influence of inflammation,and to clarify the mechanism and provide theoretical guidance of new means for treating H.pylori infection.
Aberrant miRNAs expression has also been frequently reported in various pathogenassociated tumors. Previously, we used miRNAs microarray to detect the expressionprofiles of cellular miRNAs during H. pylori infection. Noticeably, the expression level ofmiR-222was significantly up-regulated. Tumor suppressor RECK as a new target ofmiR-222, inhibits MMPs involved in breakdown of extracellular matrix (ECM), which areknown to be involved in cancer progression. Our data indicates that H. pylori may function as an initiator in the process of gastric carcinogenesis by increasing miR-222, which mayfunction as a tumor oncogene to influence the proliferation of gastric cancer cells bytargeting tumor suppressor RECK.
H. pylori can inhabit in the stomach and cause gastritis, peptic ulcer, and gastric cancer.During its long co-existence with humans, host and environmental factors consist of acomplex network to regulate the host response. Accumulating evidences have found theregulation role of miRNAs in the innate and adaptive immune response. Recent reports ofour research and other studies have highlighted the regulatory role of miRNAs in H. pyloriinfection and associated diseases. In our previous study, we first found that H. pylori wasable to increase the expression of miR-146a, and miR-146a may function as novel negativeregulators to fine-tune H. pylori-induced inflammation. However, the exact mechanism ofhow H. pylori contribute the induction of miR-146a is not clear. Hence, in current study, inlight of the pivotal role of proinflammatory cytokines and CagA, we attempted to assesswhether H. pylori related proinflammatory cytokines including IL-8, TNF-α, and IL-1β, andCagA virulence factor could contribute to the induction of miR-146a in gastric cells inresponse to H. pylori infection.
【Objectives】
1. To evaluate the role of CagA in the autophagy inhibition; to determine the effect ofautophagy inhibition on inflammation upon H. pylori infection; to explore the mechanismby CagA as an autophagy inhibitory factor.
2. To investigate the expression level of miR-222in H. pylori-infected gastric mucosaand in gastric cancer, to explore its effect on cell proliferation, and to understand itsmolecular mechanism of function as a tumor oncogene.
3. To determine whether H. pylori related proinflammatory cytokines couldcontribute to the induction of miR-146a; to examine the effect of miR-146a on the releaseof IL-8, TNF-α and IL-1β; to determine whether CagA protein is responsible for miR-146ainduction during H. pylori infection.
【Methods】
1. CagA acts as a negative regulator of autophagy in H. pylori-induced inflammatoryresponse
We dected autophagy through Western-blot, confocal laser, electron microscopy and flow cytometry techniques both in AGS cells infected with Hp-WT or Hp-△CagA and in H.pylori-infected gastric tissues; AGS cells were transfected with CagA expression plasmid, theautophagy was dected before exposure to rapamycin; by exposing the cells to autophagyinhibitors or knockdown the autophagy by siRNA silencing, ELISA was used to dectet theprotein levels of IL-8, TNF-α and IL-1β, and relative luciferase activity was dectected;Western blot were used to identify whether PI3K/Akt/mTOR involved in the mechanism byCagA as an autophagy inhibitory factor; knockdown the expression of c-Met by siRNAsilencing, western-blot, confocal laser and flow cytometry techniques were used to dectectthe autophgy level in AGS cells infected with Hp-WT or Hp-△CagA.
2. Increased miR-222in H. pylori-associated gastric cancer correlated with tumorprogression by promoting cancer cell proliferation and targeting RECK
RT-PCR analysis was ued to detect the miR-222expression in gastric epithelial cellsinfected by H. pylori or in H. pylori-infected gastric tissues; RT-PCR analysis was used todetect the miR-222expression in gastric cancer cell lines as well as gastric cancer tissues;the effect of miR-222over-expression on cell proliferation was evaluated by CCK-8assay;to further test the biological effect of miR-222on the growth of gastric cancer cells, thecolony formation assay was performed; Luciferase assay, GFP repression experiments andWestern blot were used to identify the targets of miR-222; RT-PCR analysis was used to address thecorrelation between RECK and miR-222in gastric cancer tissues; knockdown the expression ofRECK in AGS cells by siRNA silencing, CCK-8assay and colony formation assay were usedto investigate whether reduction of RECK expression may mimic the proliferation-promoting effectof miR-222overexpression.
3. H. pylori related proinflammatory cytokines contribute to the induction ofmiR-146a in human gastric epithelial cells
We stimulated HGC-27cells with IL-8, TNF-α and or IL-1β and measured theexpression of miR-146a by RT-PCR; the expression of miR-146a was assessed by RT-PCRbefore knockdown the expression of IL-8, TNF-α and IL-1β by RNA interference; wetransfected NF-κB reporter plasmid to HGC-27cells, and relative luciferase activity wasdectected before stimulation with IL-8, TNF-α and IL-1β; the expression of miR-146a byexposure to IL-8, TNF-α and IL-1β was measured by RT-PCR before pretreated withNF-κB inhibitor; when HGC-27cells were respectively transfected with miR-146a mimics, inhibitors, and miRcontrol, followed by H. pylori infection, the mRNA and protein levels ofIL-8, TNF-α and IL-1β were examined by RT-PCR and ELISA; we compared the miR-146alevel by RT-PCR in HGC-27cells infected with wild-type or cagA deficient H. pylori;HGC-27cells were transfected with CagA expression plasmid, transfection efficiency wasconfirmed by Western blot and fluorescence microscopy, and measured the expression ofmiR-146a by RT-PCR.
【Conclusions】
1. H. pylori CagA inhibits autophagy in gastric epithelial cells; Inhibition ofautophagic pathways could inhance the proinflammatory cytokine production in AGS cellsinfected with Hp-△CagA; The PI3K/Akt/mTOR signaling pathway is required for theinhibition of autophagy by CagA; c-Met is required for the inhibition of autophagy byCagA.
2. MiR-222level was up-regulated by H. pylori infection as well as in gastric cancer;Over-expression of miR-222promoted cell proliferation and colony formation; RECK as anew target of miR-222and inversely correlated with miR-222; RNA interference silencingof RECK can mimic the oncogenic effects of miR-222.
3. H. pylori related proinflammatory cytokines such as IL-8, TNF-α and IL-1β mayinduce the expression of miR-146a in gastric epithelial cells, while the induction ofmiR-146a upon H. pylori stimulation is independent of above proinflammatory cytokines;NF-κB pathway is required for the induction of miR-146a in H. pylori related cytokinesstimulation; The increase of miR-146a may play a potential role in the negative feedbackregulation of IL-8, TNF-α and IL-1β; CagA is not necessarily required for miR-146ainduction during H. pylori infection.
【Significance】
1. These results provide new insight into the effects of CagA on autophagy inhibitionduring H. pylori infection, and provide a possible intervention strategy for new treatmentof H. pylori induced inflammation.
2. Our data indicates that H. pylori may function as an initiator in the process ofgastric carcinogenesis by increasing miR-222, which may function as a tumor oncogene toinfluence the proliferation of gastric cancer cells by targeting tumor suppressor RECK.Analyses of miRNA regulation will provide opportunities to develop new means for diagnosis and treatment of gastric cancer.
3. The exact mechanism of how H. pylori contribute diseases is complex and neededto be further investigated, the discovery of miRNAs provides us a new sight. We attemptto clear the exact mechanism of how H. pylori contribute the induction of miR-146a. Thismay help us to elucidate the pathogenic mechanism of H. pylori infection and to explorenovel and effective therapies for H. pylori infection.
H. pylori的感染与致病，是一个与机体相互斗争的过程。自噬在抗病原微生物感染以及其导致的炎症中发挥重要作用。自噬受到抑制，可以促进炎性因子的释放，引起炎症反应，导致疾病的发生发展。一方面，H. pylori感染胃上皮细胞可诱导自噬,通过自噬降解病原体或其分泌的毒性分子起到保护胃上皮细胞的作用；另一方面，H.pylori为了达到感染和致病目的，存在某种机制对抗自噬。本研究提出CagA负性调控幽门螺杆菌诱导的胃上皮细胞自噬在慢性胃炎发生中的作用机制： H. pylori感染可活化胃粘膜上皮细胞内肝细胞生长因子(HGF)的受体c-Met,磷酸化c-Met可与通过Ⅳ分泌系统转运至胃上皮细胞内的CagA发生相互作用，启动了其下游的PI3K/Akt信号途径的活化,通过调节自噬通路中的信号蛋白mTOR而发挥抑制自噬的作用，从而影响到自噬对胃上皮细胞保护作用，导致CagA阳性H. pylori菌株胃上皮细胞炎症反应较重，引起慢性胃炎、消化性溃疡，甚至胃癌等疾病。
miRNAs作为一类新的癌基因或抑癌基因，其表达失衡与多种肿瘤的发生密切相关。前期研究中，我们通过芯片分析H. pylori感染胃上皮细胞miRNAs的变化，发现与肿瘤发生密切相关的miR-222表达上调，而其靶基因RECK作为一种抑癌基因，可以负性调控基质金属蛋白酶(MMP)，抑制肿瘤的生长和转移，其在胃癌组织和细胞株中表达缺失，与胃癌的预后呈负相关。因此，我们推测miR-222可能是一个与H.pylori感染和胃癌发生密切相关的癌基因。H. pylori感染能够诱导miR-222的高表达，而miR-222可能通过抑制下游抑癌基因RECK的表达，调控细胞的增殖和凋亡，促进胃癌的发生和转移。
H. pylori可以在人体特殊的复杂多变的胃环境中长期定居，其慢性持续性感染在所致疾病的发生发展中发挥着重要作用。近年来，越来越多的证据显示miRNAs在多个环节参与调控机体固有免疫应答和获得性免疫反应，在宿主抗病原微生物感染时扮演重要的角色。前期研究发现H. pylori感染相关的miR-146a通过抑制靶基因IRAK1、TRAF6和COX-2的表达，降低NF-κB的活性，从而抑制H. pylori感染导致的炎症因子的释放。为深入研究在H. pylori感染诱导miR-146a表达过程中究竟什么因素起关键作用，我们观察了H. pylori感染相关炎性细胞因子以及H. pylori毒素蛋白CagA在胃上皮细胞中对miR-146a表达的影响。
【研究目的】
1.评价H. pylori感染胃上皮细胞诱导自噬发生的过程中CagA对自噬的抑制作用；观察自噬抑制对H. pylori感染引起的炎症反应的影响；探讨CagA负性调控H. pylori诱导的胃上皮细胞自噬的机制。
2.分析H. pylori感染胃上皮细胞、胃粘膜组织以及胃癌细胞株、胃癌组织中miR-222的表达；筛选并鉴定miR-222的靶基因，探讨其致癌机制。
3.观察H. pylori相关炎性因子IL-8、TNF-α以及IL-1β刺激HGC-27细胞对miR-146a表达影响；评价miR-146a对H. pylori感染诱导炎症因子释放的影响；探讨毒力因子CagA对H. pylori感染诱导miR-146a的表达影响。
【研究方法】
1.幽门螺杆菌CagA抑制胃上皮细胞自噬及其在慢性胃炎发生中的作用机制研究
Western-blot、激光共聚焦、电镜以及流式细胞技术分别检测Hp-WT或Hp-△CagA感染人胃上皮细胞自噬模型和临床胃粘膜标本自噬水平；检测体外过表达CagA对Rapamycin诱导自噬的影响；使用化学药物法抑制自噬或利用siRNA技术阻断自噬，ELISA分别检测Hp-WT和Hp-△CagA两种菌株感染诱导炎症因子分泌的水平，荧光素酶法检测NF-κB荧光素酶报告基因活性；Western-blot检测PI3K/Akt/mTOR信号途径是否参与了H. pylori感染过程中CagA对自噬的抑制作用；利用siRNA沉默c-Met表达，Western-blot、激光共聚焦显微镜、流式细胞技术检测H. pylori感染诱导的自噬情况。
2. miR-222高表达在幽门螺杆菌感染诱发胃癌中的作用及机制研究
RT-PCR检测H. pylori感染胃上皮细胞系前后以及H. pylori感染患者胃粘膜组织、胃癌细胞株以及胃癌组织中miR-222的表达；CCK-8法检测miR-222对两种胃癌细胞株增殖的影响，分析miR-222对两种胃癌细胞株克隆形成的影响；利用双荧光素酶实验、GFP荧光抑制实验、Western blot鉴定靶基因；利用RT-PCR技术检测靶基因与miR-222胃癌组织样本中的表达水平及相关性；CCK-8法和克隆形成实验分别检测沉默RECK表达是否可以模拟过表达miR-222对胃癌细胞AGS增殖和克隆形成的影响。
3. H. pylori感染HGC-27细胞miR-146a表达调节机制初步研究
利用RT-PCR技术检测IL-8、TNF-α以及IL-1β刺激HGC-27细胞后miR-146a表达情况；siRNA沉默IL-8、TNF-α和IL-1β的表达，RT-PCR技术检测H. pylori感染后miR-146a表达；荧光素酶法检测IL-8、TNF-α或IL-1β刺激后NF-κB荧光素酶报告基因活性；分别用IL-8、TNF-α或IL-1β刺激NF-κB抑制剂(BAY-117082)处理过的HGC-27细胞，RT-PCR技术检测miR-146a表达；RT-PCR和ELISA技术分别检测过表达miR-146a对H.pylori感染炎症因子mRNA和蛋白水平； CagA阳性或阴性H. pylori菌株感染HGC-27细胞，RT-PCR技术检测miR-146a表达；转染HGC-27细胞pEGFP-C1-CagA质粒，荧光显微镜和免疫印迹检测转染效率，RT-PCR技术检测内源性高表达CagA对miR-146a的表达影响。
【主要结论】
1. CagA具有抑制H. pylori诱导胃上皮细胞自噬的作用；抑制自噬可以增强Hp-△CagA感染介导的炎症程度；CagA可能通过PI3K/Akt/mTOR信号途径介导了H. pylori感染过程中对自噬的抑制作用；c-Met分子可能参与了CagA抑制H. pylori感染胃上皮细胞自噬过程。
2.无论是体外还是体内H. pylori感染均能诱导miR-222的表达上调；miR-222在胃癌细胞株和胃癌组织中均呈高表达；RECK为miR-222的新靶基因；H. pylori感染诱导的miR-222的高表达，可能通过靶向抑制RECK蛋白的表达，来发挥促进肿瘤细胞增殖的作用；胃癌组织中，miR-222与RECK转录水平表达量呈现很好的负相关。
3. H. pylori相关炎性因子刺激胃上皮细胞HGC-27诱导miR-146a表达上调，但对于miR-146a的高表达为非必要条件； NF-κB活化参与了IL-8、 TNF-α以及IL-1β诱导miR-146a高表达；过表达miR-146a可以抑制H. pylori感染相关炎症因子的释放；H.pylori感染对miR-146a的诱导不依赖于毒素蛋白CagA。
【研究意义】
1.本研究首次提出幽门螺杆菌CagA作为一种负性调控因子，通过抑制自噬引起慢性胃炎的新观点，并在体内外从现象到机制进行了探讨，为深入理解和认识H. pylori慢性感染致病机制、寻找H. pylori感染有效防治手段提供了新的视角。
2. miR-222可能参与H. pylori感染诱发胃癌的过程，可以作为胃癌诊断或治疗的靶点，在H. pylori感染与胃癌形成的过程中发挥调节作用，研究为阐明H. pylori的致癌机制提供了新线索。
3. H. pylori感染引起的炎症免疫反应调控机制十分复杂且涉及诸多环节。miRNAs在H. pylori感染的免疫调节中发挥重要作用。本文对H. pylori感染诱导miR-146a表达的调节机制予以了初步探索，miR-146a可能成为H. pylori感染诊断、治疗和预后的新靶点。
Helicobacter pylori (H. pylori) is present in the stomach of at least half of the world’spopulation, and many studies have indicated that persistent colonization of the stomach byH. pylori causes gastric diseases such as chronic gastritis, peptic ulcer disease,mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. However, themechanism by which H. pylori induce proinflammatory responses in gastric epithelial cellshas long remained a mystery.
During H. pylori infection, host and environmental factors consist of a complex war toregulate the host response. Autophagy is thought to serve as an innate defense mechanismagainst infection. Recent studies have implicated inhibition of autophagy causes fuelinflammation. In this process, H. pylori can induce autophagy in gastric epithelial cells,which play a role in the protection of gastric epithelial cells. On the other hand, thepathogens are able to subvert autophagy as astrategy for pathopoiesis. Here, we showed thatCagA may be a negative regulated factor in autophagy induced by H. pylori. We willexplore the mechanism that H. pylori infection can activate c-Met, and then phosphorylasedc-Met interact with CagA which transport through the Ⅳ secrete system, and thenPI3K/Akt/mTOR signal way was activated to inhibit autophagy. The aim is to detect forautophagy inhibited by CagA in gastric epithelium cells and the influence of inflammation,and to clarify the mechanism and provide theoretical guidance of new means for treating H.pylori infection.
Aberrant miRNAs expression has also been frequently reported in various pathogenassociated tumors. Previously, we used miRNAs microarray to detect the expressionprofiles of cellular miRNAs during H. pylori infection. Noticeably, the expression level ofmiR-222was significantly up-regulated. Tumor suppressor RECK as a new target ofmiR-222, inhibits MMPs involved in breakdown of extracellular matrix (ECM), which areknown to be involved in cancer progression. Our data indicates that H. pylori may function as an initiator in the process of gastric carcinogenesis by increasing miR-222, which mayfunction as a tumor oncogene to influence the proliferation of gastric cancer cells bytargeting tumor suppressor RECK.
H. pylori can inhabit in the stomach and cause gastritis, peptic ulcer, and gastric cancer.During its long co-existence with humans, host and environmental factors consist of acomplex network to regulate the host response. Accumulating evidences have found theregulation role of miRNAs in the innate and adaptive immune response. Recent reports ofour research and other studies have highlighted the regulatory role of miRNAs in H. pyloriinfection and associated diseases. In our previous study, we first found that H. pylori wasable to increase the expression of miR-146a, and miR-146a may function as novel negativeregulators to fine-tune H. pylori-induced inflammation. However, the exact mechanism ofhow H. pylori contribute the induction of miR-146a is not clear. Hence, in current study, inlight of the pivotal role of proinflammatory cytokines and CagA, we attempted to assesswhether H. pylori related proinflammatory cytokines including IL-8, TNF-α, and IL-1β, andCagA virulence factor could contribute to the induction of miR-146a in gastric cells inresponse to H. pylori infection.
【Objectives】
1. To evaluate the role of CagA in the autophagy inhibition; to determine the effect ofautophagy inhibition on inflammation upon H. pylori infection; to explore the mechanismby CagA as an autophagy inhibitory factor.
2. To investigate the expression level of miR-222in H. pylori-infected gastric mucosaand in gastric cancer, to explore its effect on cell proliferation, and to understand itsmolecular mechanism of function as a tumor oncogene.
3. To determine whether H. pylori related proinflammatory cytokines couldcontribute to the induction of miR-146a; to examine the effect of miR-146a on the releaseof IL-8, TNF-α and IL-1β; to determine whether CagA protein is responsible for miR-146ainduction during H. pylori infection.
【Methods】
1. CagA acts as a negative regulator of autophagy in H. pylori-induced inflammatoryresponse
We dected autophagy through Western-blot, confocal laser, electron microscopy and flow cytometry techniques both in AGS cells infected with Hp-WT or Hp-△CagA and in H.pylori-infected gastric tissues; AGS cells were transfected with CagA expression plasmid, theautophagy was dected before exposure to rapamycin; by exposing the cells to autophagyinhibitors or knockdown the autophagy by siRNA silencing, ELISA was used to dectet theprotein levels of IL-8, TNF-α and IL-1β, and relative luciferase activity was dectected;Western blot were used to identify whether PI3K/Akt/mTOR involved in the mechanism byCagA as an autophagy inhibitory factor; knockdown the expression of c-Met by siRNAsilencing, western-blot, confocal laser and flow cytometry techniques were used to dectectthe autophgy level in AGS cells infected with Hp-WT or Hp-△CagA.
2. Increased miR-222in H. pylori-associated gastric cancer correlated with tumorprogression by promoting cancer cell proliferation and targeting RECK
RT-PCR analysis was ued to detect the miR-222expression in gastric epithelial cellsinfected by H. pylori or in H. pylori-infected gastric tissues; RT-PCR analysis was used todetect the miR-222expression in gastric cancer cell lines as well as gastric cancer tissues;the effect of miR-222over-expression on cell proliferation was evaluated by CCK-8assay;to further test the biological effect of miR-222on the growth of gastric cancer cells, thecolony formation assay was performed; Luciferase assay, GFP repression experiments andWestern blot were used to identify the targets of miR-222; RT-PCR analysis was used to address thecorrelation between RECK and miR-222in gastric cancer tissues; knockdown the expression ofRECK in AGS cells by siRNA silencing, CCK-8assay and colony formation assay were usedto investigate whether reduction of RECK expression may mimic the proliferation-promoting effectof miR-222overexpression.
3. H. pylori related proinflammatory cytokines contribute to the induction ofmiR-146a in human gastric epithelial cells
We stimulated HGC-27cells with IL-8, TNF-α and or IL-1β and measured theexpression of miR-146a by RT-PCR; the expression of miR-146a was assessed by RT-PCRbefore knockdown the expression of IL-8, TNF-α and IL-1β by RNA interference; wetransfected NF-κB reporter plasmid to HGC-27cells, and relative luciferase activity wasdectected before stimulation with IL-8, TNF-α and IL-1β; the expression of miR-146a byexposure to IL-8, TNF-α and IL-1β was measured by RT-PCR before pretreated withNF-κB inhibitor; when HGC-27cells were respectively transfected with miR-146a mimics, inhibitors, and miRcontrol, followed by H. pylori infection, the mRNA and protein levels ofIL-8, TNF-α and IL-1β were examined by RT-PCR and ELISA; we compared the miR-146alevel by RT-PCR in HGC-27cells infected with wild-type or cagA deficient H. pylori;HGC-27cells were transfected with CagA expression plasmid, transfection efficiency wasconfirmed by Western blot and fluorescence microscopy, and measured the expression ofmiR-146a by RT-PCR.
【Conclusions】
1. H. pylori CagA inhibits autophagy in gastric epithelial cells; Inhibition ofautophagic pathways could inhance the proinflammatory cytokine production in AGS cellsinfected with Hp-△CagA; The PI3K/Akt/mTOR signaling pathway is required for theinhibition of autophagy by CagA; c-Met is required for the inhibition of autophagy byCagA.
2. MiR-222level was up-regulated by H. pylori infection as well as in gastric cancer;Over-expression of miR-222promoted cell proliferation and colony formation; RECK as anew target of miR-222and inversely correlated with miR-222; RNA interference silencingof RECK can mimic the oncogenic effects of miR-222.
3. H. pylori related proinflammatory cytokines such as IL-8, TNF-α and IL-1β mayinduce the expression of miR-146a in gastric epithelial cells, while the induction ofmiR-146a upon H. pylori stimulation is independent of above proinflammatory cytokines;NF-κB pathway is required for the induction of miR-146a in H. pylori related cytokinesstimulation; The increase of miR-146a may play a potential role in the negative feedbackregulation of IL-8, TNF-α and IL-1β; CagA is not necessarily required for miR-146ainduction during H. pylori infection.
【Significance】
1. These results provide new insight into the effects of CagA on autophagy inhibitionduring H. pylori infection, and provide a possible intervention strategy for new treatmentof H. pylori induced inflammation.
2. Our data indicates that H. pylori may function as an initiator in the process ofgastric carcinogenesis by increasing miR-222, which may function as a tumor oncogene toinfluence the proliferation of gastric cancer cells by targeting tumor suppressor RECK.Analyses of miRNA regulation will provide opportunities to develop new means for diagnosis and treatment of gastric cancer.
3. The exact mechanism of how H. pylori contribute diseases is complex and neededto be further investigated, the discovery of miRNAs provides us a new sight. We attemptto clear the exact mechanism of how H. pylori contribute the induction of miR-146a. Thismay help us to elucidate the pathogenic mechanism of H. pylori infection and to explorenovel and effective therapies for H. pylori infection.
引文
[1] Algood HM&Cover TL (2006) Helicobacter pylori persistence: an overview ofinteractions between H. pylori and host immune defenses. Clin Microbiol Rev19(4):597-613
[2] Backert S, et al.(2004) Functional analysis of the cag pathogenicity island inHelicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastriccancer. Infection and immunity72(2):1043-1056
[3] Nomura A, et al.(1991) Helicobacter pylori infection and gastric carcinoma amongJapanese Americans in Hawaii. The New England journal of medicine325(16):1132-1136
[4] Pan ZJ, et al.(1997) Equally high prevalences of infection with cagA-positiveHelicobacter pylori in Chinese patients with peptic ulcer disease and those withchronic gastritis-associated dyspepsia. Journal of clinical microbiology35(6):1344-1347
[5] Odenbreit S, et al.(2000) Translocation of Helicobacter pylori CagA into gastricepithelial cells by type IV secretion. Science287(5457):1497-1500
[6] Suzuki M, et al.(2011) Attenuated CagA oncoprotein in Helicobacter pylori fromAmerindians in Peruvian Amazon. The Journal of biological chemistry286(34):29964-29972
[7] Levine B&Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell132(1):27-42
[8] Boya P, et al.(2005) Inhibition of macroautophagy triggers apoptosis. Molecular andcellular biology25(3):1025-1040
[9] Ogawa M&Sasakawa C (2006) Bacterial evasion of the autophagic defense system.Current opinion in microbiology9(1):62-68
[10] Birmingham CL, et al.(2007) Listeria monocytogenes evades killing by autophagyduring colonization of host cells. Autophagy3(5):442-451
[11] Schnaith A, et al.(2007) Staphylococcus aureus subvert autophagy for induction ofcaspase-independent host cell death. The Journal of biological chemistry282(4):2695-2706
[12] Hernandez LD, Pypaert M, Flavell RA,&Galan JE (2003) A Salmonella proteincauses macrophage cell death by inducing autophagy. The Journal of cell biology163(5):1123-1131
[13] Nakagawa I, et al.(2004) Autophagy defends cells against invading group AStreptococcus. Science306(5698):1037-1040
[14] Moscat J&Diaz-Meco MT (2009) p62at the crossroads of autophagy, apoptosis, andcancer. Cell137(6):1001-1004
[15] Lee HM, et al.(2011) Autophagy negatively regulates keratinocyte inflammatoryresponses via scaffolding protein p62/SQSTM1. J Immunol186(2):1248-1258
[16] Saitoh T, et al.(2008) Loss of the autophagy protein Atg16L1enhances endotoxin-induced IL-1beta production. Nature456(7219):264-268
[17] Tabassam FH, Graham DY,&Yamaoka Y (2009) Helicobacter pylori activateepidermal growth factor receptor-and phosphatidylinositol3-OH kinase-dependentAkt and glycogen synthase kinase3beta phosphorylation. Cellular microbiology11(1):70-82
[18] Meijer AJ&Codogno P (2006) Signalling and autophagy regulation in health, agingand disease. Molecular aspects of medicine27(5-6):411-425
[19] Backert S, Tegtmeyer N,&Selbach M (2010) The versatility of Helicobacter pyloriCagA effector protein functions: The master key hypothesis. Helicobacter15(3):163-176
[20] Kwok T, et al.(2007) Helicobacter exploits integrin for type IV secretion and kinaseactivation. Nature449(7164):862-866
[21] Suzuki M, et al.(2009) Helicobacter pylori CagA phosphorylation-independentfunction in epithelial proliferation and inflammation. Cell Host Microbe5(1):23-34
[22] Mizushima N, Yoshimori T,&Levine B (2010) Methods in mammalian autophagyresearch. Cell140(3):313-326
[23] Gutierrez MG, et al.(2004) Autophagy is a defense mechanism inhibiting BCG andMycobacterium tuberculosis survival in infected macrophages. Cell119(6):753-766
[24] Deretic V&Levine B (2009) Autophagy, immunity, and microbial adaptations. CellHost Microbe5(6):527-549
[25] Choy A, et al.(2012) The Legionella effector RavZ inhibits host autophagy throughirreversible Atg8deconjugation. Science338(6110):1072-1076
[26] Chaumorcel M, Souquere S, Pierron G, Codogno P,&Esclatine A (2008) Humancytomegalovirus controls a new autophagy-dependent cellular antiviral defensemechanism. Autophagy4(1):46-53
[27] Terebiznik MR, et al.(2009) Effect of Helicobacter pylori's vacuolating cytotoxin onthe autophagy pathway in gastric epithelial cells. Autophagy5(3):370-379
[28] Tang B, et al.(2012) Compromised autophagy by MIR30B benefits the intracellularsurvival of Helicobacter pylori. Autophagy8(7):1045-1057
[29] Harris J, et al.(2011) Autophagy controls IL-1beta secretion by targeting pro-IL-1betafor degradation. The Journal of biological chemistry286(11):9587-9597
[30] Fujishima Y, et al.(2011) Autophagy in the intestinal epithelium reducesendotoxin-induced inflammatory responses by inhibiting NF-kappaB activation.Archives of biochemistry and biophysics506(2):223-235
[31] Hirata Y, et al.(2006) Helicobacter pylori induces IkappaB kinase alpha nucleartranslocation and chemokine production in gastric epithelial cells. Infect Immun74(3):1452-1461
[32] Lamb A, et al.(2009) Helicobacter pylori CagA activates NF-kappaB by targetingTAK1for TRAF6-mediated Lys63ubiquitination. EMBO Rep10(11):1242-1249
[33] Anonymous (1994) IARC working group on the evaluation of carcinogenic risks tohumans: some industrial chemicals. Lyon,15-22February1994. IARC Monogr EvalCarcinog Risks Hum60:1-560
[34] Bornschein J, Rokkas T, Selgrad M,&Malfertheiner P (2009) Helicobacter pyloriand clinical aspects of gastric cancer. Helicobacter14Suppl1:41-45
[35] Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell136(2):215-233
[36] Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer.Nat Rev Genet10(10):704-714
[37] Tavazoie SF, et al.(2008) Endogenous human microRNAs that suppress breast cancermetastasis. Nature451(7175):147-152
[38] Meng F, et al.(2007) MicroRNA-21regulates expression of the PTEN tumorsuppressor gene in human hepatocellular cancer. Gastroenterology133(2):647-658
[39] Bandi N, et al.(2009) miR-15a and miR-16are implicated in cell cycle regulation in aRb-dependent manner and are frequently deleted or down-regulated in non-small celllung cancer. Cancer Res69(13):5553-5559
[40] Petrocca F, et al.(2008) E2F1-regulated microRNAs impair TGFbeta-dependentcell-cycle arrest and apoptosis in gastric cancer. Cancer Cell13(3):272-286
[41] Ueda T, et al.(2010) Relation between microRNA expression and progression andprognosis of gastric cancer: a microRNA expression analysis. Lancet Oncol11(2):136-146
[42] Liu T, Tang H, Lang Y, Liu M,&Li X (2009) MicroRNA-27a functions as anoncogene in gastric adenocarcinoma by targeting prohibitin. Cancer Lett273(2):233-242
[43] Ding L, et al.(2010) MiR-375frequently downregulated in gastric cancer inhibits cellproliferation by targeting JAK2. Cell Res20(7):784-793
[44] Zhao X, et al.(2012) MicroRNA-7functions as an anti-metastatic microRNA ingastric cancer by targeting insulin-like growth factor-1receptor. Oncogene
[45] Xiao B, et al.(2009) Induction of microRNA-155during Helicobacter pyloriinfection and its negative regulatory role in the inflammatory response. J Infect Dis200(6):916-925
[46] Zhang Z, et al.(2008) miR-21plays a pivotal role in gastric cancer pathogenesis andprogression. Lab Invest88(12):1358-1366
[47] Gao C, et al.(2010) Reduced microRNA-218expression is associated with highnuclear factor kappa B activation in gastric cancer. Cancer116(1):41-49
[48] Pallante P, et al.(2006) MicroRNA deregulation in human thyroid papillarycarcinomas. Endocr Relat Cancer13(2):497-508
[49] Wong QW, et al.(2010) MiR-222overexpression confers cell migratory advantagesin hepatocellular carcinoma through enhancing AKT signaling. Clin Cancer Res16(3):867-875
[50] Felicetti F, Errico MC, Segnalini P, Mattia G,&Care A (2008) MicroRNA-221and-222pathway controls melanoma progression. Expert Rev Anticancer Ther8(11):1759-1765
[51] Hutzinger R, Mrazek J, Vorwerk S,&Huttenhofer A (2010) NcRNA-microchipanalysis: a novel approach to identify differential expression of noncoding RNAs.RNA Biol7(5):586-595
[52] Kim YK, et al.(2009) Functional links between clustered microRNAs: suppression ofcell-cycle inhibitors by microRNA clusters in gastric cancer. Nucleic Acids Res37(5):1672-1681
[53] Zhang C, et al.(2009) Co-suppression of miR-221/222cluster suppresses humanglioma cell growth by targeting p27kip1in vitro and in vivo. Int J Oncol34(6):1653-1660
[54] Zhao JJ, et al.(2008) MicroRNA-221/222negatively regulates estrogen receptoralpha and is associated with tamoxifen resistance in breast cancer. J Biol Chem283(45):31079-31086
[55] Miller TE, et al.(2008) MicroRNA-221/222confers tamoxifen resistance in breastcancer by targeting p27Kip1. J Biol Chem283(44):29897-29903
[56] Terasawa K, Ichimura A, Sato F, Shimizu K,&Tsujimoto G (2009) Sustainedactivation of ERK1/2by NGF induces microRNA-221and222in PC12cells. FEBS J276(12):3269-3276
[57] Liu X, et al.(2009) MicroRNA-222regulates cell invasion by targeting matrixmetalloproteinase1(MMP1) and manganese superoxide dismutase2(SOD2) intongue squamous cell carcinoma cell lines. Cancer Genomics Proteomics6(3):131-139
[58] Oh J, et al.(2001) The membrane-anchored MMP inhibitor RECK is a key regulatorof extracellular matrix integrity and angiogenesis. Cell107(6):789-800
[59] Song SY, Son HJ, Nam E, Rhee JC,&Park C (2006) Expression ofreversion-inducing-cysteine-rich protein with Kazal motifs (RECK) as a prognosticindicator in gastric cancer. Eur J Cancer42(1):101-108
[60] Garofalo M, Quintavalle C, Romano G, Croce CM,&Condorelli G (2012)miR221/222in cancer: their role in tumor progression and response to therapy. CurrMol Med12(1):27-33
[61] Chun-Zhi Z, et al.(2010) MicroRNA-221and microRNA-222regulate gastriccarcinoma cell proliferation and radioresistance by targeting PTEN. BMC Cancer10:367
[62] le Sage C, et al.(2007) Regulation of the p27(Kip1) tumor suppressor by miR-221and miR-222promotes cancer cell proliferation. EMBO J26(15):3699-3708
[63] Visone R, et al.(2007) MicroRNAs (miR)-221and miR-222, both overexpressed inhuman thyroid papillary carcinomas, regulate p27Kip1protein levels and cell cycle.Endocr Relat Cancer14(3):791-798
[64] Fu X, et al.(2011) Clinical significance of miR-221and its inverse correlation withp27Kip(1) in hepatocellular carcinoma. Mol Biol Rep38(5):3029-3035
[65] Garofalo M, et al.(2008) MicroRNA signatures of TRAIL resistance in humannon-small cell lung cancer. Oncogene27(27):3845-3855
[66] Fornari F, et al.(2008) MiR-221controls CDKN1C/p57and CDKN1B/p27expression in human hepatocellular carcinoma. Oncogene27(43):5651-5661
[67] Zhang C, et al.(2010) PUMA is a novel target of miR-221/222in human epithelialcancers. Int J Oncol37(6):1621-1626
[68] Di Leva G, et al.(2010) MicroRNA cluster221-222and estrogen receptor alphainteractions in breast cancer. J Natl Cancer Inst102(10):706-721
[69] Garofalo M, et al.(2009) miR-221&222regulate TRAIL resistance and enhancetumorigenicity through PTEN and TIMP3downregulation. Cancer Cell16(6):498-509
[70] Pineau P, et al.(2010) miR-221overexpression contributes to liver tumorigenesis.Proc Natl Acad Sci U S A107(1):264-269
[71] Felli N, et al.(2005) MicroRNAs221and222inhibit normal erythropoiesis anderythroleukemic cell growth via kit receptor down-modulation. Proc Natl Acad Sci US A102(50):18081-18086
[72] Takahashi C, et al.(1998) Regulation of matrix metalloproteinase-9and inhibition oftumor invasion by the membrane-anchored glycoprotein RECK. Proc Natl Acad Sci US A95(22):13221-13226
[73] Clark JC, Thomas DM, Choong PF,&Dass CR (2007) RECK--a newly discoveredinhibitor of metastasis with prognostic significance in multiple forms of cancer.Cancer Metastasis Rev26(3-4):675-683
[74] Teng GG, et al.(2013) Let-7b Is Involved in the Inflammation and ImmuneResponses Associated with Helicobacter pylori Infection by Targeting Toll-LikeReceptor4. PLoS One8(2):e56709
[75] Zabaleta J (2012) MicroRNA: A Bridge from H. pylori Infection to Gastritis andGastric Cancer Development. Front Genet3:294
[76] Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell116(2):281-297
[77] Ambros V (2004) The functions of animal microRNAs. Nature431(7006):350-355
[78] Taganov KD, Boldin MP,&Baltimore D (2007) MicroRNAs and immunity: tinyplayers in a big field. Immunity26(2):133-137
[79] Xiao C&Rajewsky K (2009) MicroRNA control in the immune system: basicprinciples. Cell136(1):26-36
[80] Gottwein E&Cullen BR (2008) Viral and cellular microRNAs as determinants ofviral pathogenesis and immunity. Cell Host Microbe3(6):375-387
[81] Jopling CL, Yi M, Lancaster AM, Lemon SM,&Sarnow P (2005) Modulation ofhepatitis C virus RNA abundance by a liver-specific MicroRNA. Science309(5740):1577-1581
[82] Jopling CL (2008) Regulation of hepatitis C virus by microRNA-122. Biochem SocTrans36(Pt6):1220-1223
[83] Chen XM, Splinter PL, O'Hara SP,&LaRusso NF (2007) A cellular micro-RNA,let-7i, regulates Toll-like receptor4expression and contributes to cholangiocyteimmune responses against Cryptosporidium parvum infection. J Biol Chem282(39):28929-28938
[84] Liu Z, et al.(2010) Up-regulated microRNA-146a negatively modulate Helicobacterpylori-induced inflammatory response in human gastric epithelial cells. MicrobesInfect12(11):854-863
[85] Yoshida N, et al.(1993) Mechanisms involved in Helicobacter pylori-inducedinflammation. Gastroenterology105(5):1431-1440
[86] Mooney C, et al.(1991) Neutrophil activation by Helicobacter pylori. Gut32(8):853-857
[87] Takashima M, Furuta T, Hanai H, Sugimura H,&Kaneko E (2001) Effects ofHelicobacter pylori infection on gastric acid secretion and serum gastrin levels inMongolian gerbils. Gut48(6):765-773
[88] Perry MM, et al.(2008) Rapid changes in microRNA-146a expression negativelyregulate the IL-1beta-induced inflammatory response in human lung alveolarepithelial cells. J Immunol180(8):5689-5698
[89] Li J, et al.(2010) Altered microRNA expression profile with miR-146a upregulationin CD4+T cells from patients with rheumatoid arthritis. Arthritis Res Ther12(3):R81
[90] Brandt S, Kwok T, Hartig R, Konig W,&Backert S (2005) NF-kappaB activation andpotentiation of proinflammatory responses by the Helicobacter pylori CagA protein.Proc Natl Acad Sci U S A102(26):9300-9305
[91] Hou J, et al.(2009) MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFNproduction in macrophages by targeting TRAF6, IRAK1, and IRAK2. J Immunol183(3):2150-2158
[92] Dai R, et al.(2008) Suppression of LPS-induced Interferon-gamma and nitric oxide insplenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism ofimmune modulation. Blood112(12):4591-4597
[93] Nahid MA, Pauley KM, Satoh M,&Chan EK (2009) miR-146a is critical forendotoxin-induced tolerance: IMPLICATION IN INNATE IMMUNITY. J Biol Chem284(50):34590-34599
[94] Bhaumik D, et al.(2008) Expression of microRNA-146suppresses NF-kappaBactivity with reduction of metastatic potential in breast cancer cells. Oncogene27(42):5643-5647
[95] Fassi Fehri L, et al.(2010) Helicobacter pylori induces miR-155in T cells in acAMP-Foxp3-dependent manner. PLoS One5(3):e9500
[1] Taganov KD, Boldin MP, Baltimore D, et al. MicroRNAs and immunity: tinyplayers in a big field [J]. Immunity.2007,26(2):133-137.
[2] Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, Lim LP, et al. The widespreadimpact of mammalian microRNAs on mRNA repression and evolution. Science2005;310:1817–21.
[3] Zhou F, Ferguson J, Chang JT. Inter-and intra-combinatorial regulation bytranscription factors and microRNAs. BMC Genomics2007;8:396.
[4] Sylvestre Y, De Guire V, Querido E, Mukhopadhyay UK, Bourdeau V, Major F, et al.An E2F/miR-20a autoregulatory feedback loop. J Biol Chem2007;282:2135–43.
[5] Espel E. The role of the AU-rich elements of mRNAs in controlling translation. SeminCell Dev Biol2005;16:59–67.
[6] Bakheet T, Frevel M, Williams BR, Greer W, Khabar KS. ARED: human AU-richelement-containing mRNA database reveals an unexpectedly diverse functionalrepertoire of encoded proteins. Nucleic Acids Res2001;29:246–54.
[7] Zubiaga AM, Belasco JG, Greenberg ME. The nonamer UUAUUUAUU is the keyAU-rich sequence motif that mediates mRNA degradation. Mol CellBiol1995;4:2219–30.
[8] Cao H, Deterding LJ, Blackshear PJ. Phosphorylation site analysis of theanti-inflammatory and mRNA-destabilizing protein tristetraprolin. Expert RevProteomics2007;4:711–26.
[9] Peng SS, Chen CY, Xu N, Shyu AB. RNA stabilization by the AU-rich elementbinding protein, HuR, an ELAV protein. EMBO J1998;17:3461–70.
[10] Loflin P, Chen CY, Shyu AB. Unraveling a cytoplasmic role for hnRNP D in the invivo mRNA destabilization directed by the AU-rich element. Genes Dev1999;13:1884–97.
[11] Lai WS, Blackshear PJ. Interactions of CCCH zinc finger proteins with mRNA:tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur inthe absence of a poly [A] tail. J Biol Chem2001;276:23144–54.
[12] Moschos SA, Williams AE, Perry MM, Birrell MA, Belvisi MG, Lindsay MA.Expression profiling in vivo demonstrates rapid changes in lung microRNA levelsfollowing lipopolysaccharide-induced inflammation but not in the anti-inflammatoryaction of glucocorticoids. BMC Genomics2007;8:240.
[13] Tili E, Michaille J-J, Cimino A, et al. Modulation of miR-155and miR-125b levelsfollowing lipopolysaccharide/TNF-αstimulation and their possible roles in regulatingthe response to endotoxin shock. J Immunol,2007,179(8):5082～5089
[14] Cameron J E, Yin Q Y, Fewell C, et al. The Epstein-Barr Virus latent membraneprotein1(LMP1) induces cellular microRNA-146a, a modulator of lymphocytesignaling pathways. J Virol,2008,82(4):1946～1958.
[15] Perry MM, Moschos SA, Williams AE, et al. Rapid changes in microRNA-146aexpression negatively regulate the IL-1β induced inflammatory response in humanlung alveolar epithelial cells［J］. J Immunol,2008,180(8):5689-5698.
[16] Jing Q, Huang S, Guth S, et al. Involvement of microRNA in AU-richelement-mediated mRNA instability. Cell,2005,120(5):623～634.
[17] Vasudevan S, Tong Y C, Steitz J A. Switching from repression to activation:microRNAs can up-regulate translation. Science,2007,318(5858):1931～1934.
[1] Deretic, V.(2009). Multiple regulatory and effector roles of autophagy in immunity.Curr Opin Immunol21,53-62.
[2] Mizushima, N., Yoshimori, T. and Levine, B.(2010). Methods in mammalianautophagy research. Cell140,313-26.
[3] Levine, B., Mizushima, N. and Virgin, H.W.(2011). Autophagy in immunity andinflammation. Nature469,323-35.
[4] Yang, Z. and Klionsky, D.J.(2010). Mammalian autophagy: core molecular machineryand signaling regulation. Curr Opin Cell Biol22,124-31.
[5] Salgame, P.(2005). Host innate and Th1responses and the bacterial factors thatcontrol Mycobacterium tuberculosis infection. Curr Opin Immunol17,374-80.
[6] Gutierrez, M.G., Master, S.S., Singh, S.B., Taylor, G.A., Colombo, M.I. and Deretic, V.(2004). Autophagy is a defense mechanism inhibiting BCG and Mycobacteriumtuberculosis survival in infected macrophages. Cell119,753-66.
[7] Singh, S.B., Davis, A.S., Taylor, G.A. and Deretic, V.(2006). Human IRGM inducesautophagy to eliminate intracellular mycobacteria. Science313,1438-41.
[8] Harris, J., De Haro, S.A., Master, S.S., Keane, J., Roberts, E.A., Delgado, M. andDeretic, V.(2007). T helper2cytokines inhibit autophagic control of intracellularMycobacterium tuberculosis. Immunity27,505-17.
[9] Andrade, R.M., Wessendarp, M., Gubbels, M.J., Striepen, B. and Subauste, C.S.(2006). CD40induces macrophage anti-Toxoplasma gondii activity by triggeringautophagy-dependent fusion of pathogen-containing vacuoles and lysosomes. J ClinInvest116,2366-77.
[10] Baregamian, N., Song, J., Bailey, C.E., Papaconstantinou, J., Evers, B.M. and Chung,D.H.(2009). Tumor necrosis factor-alpha and apoptosis signal-regulating kinase1control reactive oxygen species release, mitochondrial autophagy, and c-JunN-terminal kinase/p38phosphorylation during necrotizing enterocolitis. Oxid MedCell Longev2,297-306.
[11] Harris, J. and Keane, J.(2010). How tumour necrosis factor blockers interfere withtuberculosis immunity. Clin Exp Immunol161,1-9.
[12] Jia, G., Cheng, G., Gangahar, D.M. and Agrawal, D.K.(2006). Insulin-like growthfactor-1and TNF-alpha regulate autophagy through c-jun N-terminal kinase and Aktpathways in human atherosclerotic vascular smooth cells. Immunol Cell Biol84,448-54.
[13] Jia, L., Dourmashkin, R.R., Allen, P.D., Gray, A.B., Newland, A.C. and Kelsey, S.M.(1997). Inhibition of autophagy abrogates tumour necrosis factor alpha inducedapoptosis in human T-lymphoblastic leukaemic cells. Br J Haematol98,673-85.
[14] Keller, C.W., Fokken, C., Turville, S.G., Lunemann, A., Schmidt, J., Munz, C. andLunemann, J.D.(2011). TNF-alpha induces macroautophagy and regulates MHC classII expression in human skeletal muscle cells. J Biol Chem286,3970-80.
[15] Ling, Y.M., Shaw, M.H., Ayala, C., Coppens, I., Taylor, G.A., Ferguson, D.J. and Yap,G.S.(2006). Vacuolar and plasma membrane stripping and autophagic elimination ofToxoplasma gondii in primed effector macrophages. J Exp Med203,2063-71.
[16] Sivaprasad, U. and Basu, A.(2008). Inhibition of ERK attenuates autophagy andpotentiates tumour necrosis factor-alpha-induced cell death in MCF-7cells. J Cell MolMed12,1265-71.
[17] Djavaheri-Mergny, M., Amelotti, M., Mathieu, J., Besancon, F., Bauvy, C., Souquere,S., Pierron, G. and Codogno, P.(2006). NF-kappaB activation represses tumor necrosisfactor-alpha-induced autophagy. J Biol Chem281,30373-82.
[18] Arico, S., Petiot, A., Bauvy, C., Dubbelhuis, P.F., Meijer, A.J., Codogno, P. andOgier-Denis, E.(2001). The tumor suppressor PTEN positively regulatesmacroautophagy by inhibiting the phosphatidylinositol3-kinase/protein kinase Bpathway. J Biol Chem276,35243-6.
[19] Van Grol, J., Subauste, C., Andrade, R.M., Fujinaga, K., Nelson, J. and Subauste, C.S.(2010). HIV-1inhibits autophagy in bystander macrophage/monocytic cells throughSrc-Akt and STAT3. PLoS One5, e11733.
[20] Ni Cheallaigh, C., Keane, J., Lavelle, E.C., Hope, J.C. and Harris, J.(2011).Autophagy in the immune response to tuberculosis: clinical perspectives. Clin ExpImmunol164,291-300.
[21] Kiyono, K., Suzuki, H.I., Matsuyama, H., Morishita, Y., Komuro, A., Kano, M.R.,Sugimoto, K. and Miyazono, K.(2009). Autophagy is activated by TGF-beta andpotentiates TGF-beta-mediated growth inhibition in human hepatocellular carcinomacells. Cancer Res69,8844-52.
[22] Roca, H., Varsos, Z.S., Sud, S., Craig, M.J., Ying, C. and Pienta, K.J.(2009). CCL2and interleukin-6promote survival of human CD11b+peripheral blood mononuclearcells and induce M2-type macrophage polarization. J Biol Chem284,34342-54.
[23] Harris, J. et al.(2011). Autophagy controls IL-1beta secretion by targetingpro-IL-1beta for degradation. J Biol Chem286,9587-97.
[24] Fujishima, Y. et al.(2011). Autophagy in the intestinal epithelium reducesendotoxin-induced inflammatory responses by inhibiting NF-kappaB activation. ArchBiochem Biophys506,223-35.
[25] Li, Y.Y. et al.(2011). Autophagy is required for toll-like receptor-mediatedinterleukin-8production in intestinal epithelial cells. Int J Mol Med27,337-44.
[26] Lee, H.K., Lund, J.M., Ramanathan, B., Mizushima, N. and Iwasaki, A.(2007).Autophagy-dependent viral recognition by plasmacytoid dendritic cells. Science315,1398-401.
[27] Tal, M.C., Sasai, M., Lee, H.K., Yordy, B., Shadel, G.S. and Iwasaki, A.(2009).Absence of autophagy results in reactive oxygen species-dependent amplification ofRLR signaling. Proc Natl Acad Sci U S A106,2770-5.
[28] Saitoh, T. et al.(2008). Loss of the autophagy protein Atg16L1enhancesendotoxin-induced IL-1beta production. Nature456,264-8.
[2] Backert S, et al.(2004) Functional analysis of the cag pathogenicity island inHelicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastriccancer. Infection and immunity72(2):1043-1056
[3] Nomura A, et al.(1991) Helicobacter pylori infection and gastric carcinoma amongJapanese Americans in Hawaii. The New England journal of medicine325(16):1132-1136
[4] Pan ZJ, et al.(1997) Equally high prevalences of infection with cagA-positiveHelicobacter pylori in Chinese patients with peptic ulcer disease and those withchronic gastritis-associated dyspepsia. Journal of clinical microbiology35(6):1344-1347
[5] Odenbreit S, et al.(2000) Translocation of Helicobacter pylori CagA into gastricepithelial cells by type IV secretion. Science287(5457):1497-1500
[6] Suzuki M, et al.(2011) Attenuated CagA oncoprotein in Helicobacter pylori fromAmerindians in Peruvian Amazon. The Journal of biological chemistry286(34):29964-29972
[7] Levine B&Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell132(1):27-42
[8] Boya P, et al.(2005) Inhibition of macroautophagy triggers apoptosis. Molecular andcellular biology25(3):1025-1040
[9] Ogawa M&Sasakawa C (2006) Bacterial evasion of the autophagic defense system.Current opinion in microbiology9(1):62-68
[10] Birmingham CL, et al.(2007) Listeria monocytogenes evades killing by autophagyduring colonization of host cells. Autophagy3(5):442-451
[11] Schnaith A, et al.(2007) Staphylococcus aureus subvert autophagy for induction ofcaspase-independent host cell death. The Journal of biological chemistry282(4):2695-2706
[12] Hernandez LD, Pypaert M, Flavell RA,&Galan JE (2003) A Salmonella proteincauses macrophage cell death by inducing autophagy. The Journal of cell biology163(5):1123-1131
[13] Nakagawa I, et al.(2004) Autophagy defends cells against invading group AStreptococcus. Science306(5698):1037-1040
[14] Moscat J&Diaz-Meco MT (2009) p62at the crossroads of autophagy, apoptosis, andcancer. Cell137(6):1001-1004
[15] Lee HM, et al.(2011) Autophagy negatively regulates keratinocyte inflammatoryresponses via scaffolding protein p62/SQSTM1. J Immunol186(2):1248-1258
[16] Saitoh T, et al.(2008) Loss of the autophagy protein Atg16L1enhances endotoxin-induced IL-1beta production. Nature456(7219):264-268
[17] Tabassam FH, Graham DY,&Yamaoka Y (2009) Helicobacter pylori activateepidermal growth factor receptor-and phosphatidylinositol3-OH kinase-dependentAkt and glycogen synthase kinase3beta phosphorylation. Cellular microbiology11(1):70-82
[18] Meijer AJ&Codogno P (2006) Signalling and autophagy regulation in health, agingand disease. Molecular aspects of medicine27(5-6):411-425
[19] Backert S, Tegtmeyer N,&Selbach M (2010) The versatility of Helicobacter pyloriCagA effector protein functions: The master key hypothesis. Helicobacter15(3):163-176
[20] Kwok T, et al.(2007) Helicobacter exploits integrin for type IV secretion and kinaseactivation. Nature449(7164):862-866
[21] Suzuki M, et al.(2009) Helicobacter pylori CagA phosphorylation-independentfunction in epithelial proliferation and inflammation. Cell Host Microbe5(1):23-34
[22] Mizushima N, Yoshimori T,&Levine B (2010) Methods in mammalian autophagyresearch. Cell140(3):313-326
[23] Gutierrez MG, et al.(2004) Autophagy is a defense mechanism inhibiting BCG andMycobacterium tuberculosis survival in infected macrophages. Cell119(6):753-766
[24] Deretic V&Levine B (2009) Autophagy, immunity, and microbial adaptations. CellHost Microbe5(6):527-549
[25] Choy A, et al.(2012) The Legionella effector RavZ inhibits host autophagy throughirreversible Atg8deconjugation. Science338(6110):1072-1076
[26] Chaumorcel M, Souquere S, Pierron G, Codogno P,&Esclatine A (2008) Humancytomegalovirus controls a new autophagy-dependent cellular antiviral defensemechanism. Autophagy4(1):46-53
[27] Terebiznik MR, et al.(2009) Effect of Helicobacter pylori's vacuolating cytotoxin onthe autophagy pathway in gastric epithelial cells. Autophagy5(3):370-379
[28] Tang B, et al.(2012) Compromised autophagy by MIR30B benefits the intracellularsurvival of Helicobacter pylori. Autophagy8(7):1045-1057
[29] Harris J, et al.(2011) Autophagy controls IL-1beta secretion by targeting pro-IL-1betafor degradation. The Journal of biological chemistry286(11):9587-9597
[30] Fujishima Y, et al.(2011) Autophagy in the intestinal epithelium reducesendotoxin-induced inflammatory responses by inhibiting NF-kappaB activation.Archives of biochemistry and biophysics506(2):223-235
[31] Hirata Y, et al.(2006) Helicobacter pylori induces IkappaB kinase alpha nucleartranslocation and chemokine production in gastric epithelial cells. Infect Immun74(3):1452-1461
[32] Lamb A, et al.(2009) Helicobacter pylori CagA activates NF-kappaB by targetingTAK1for TRAF6-mediated Lys63ubiquitination. EMBO Rep10(11):1242-1249
[33] Anonymous (1994) IARC working group on the evaluation of carcinogenic risks tohumans: some industrial chemicals. Lyon,15-22February1994. IARC Monogr EvalCarcinog Risks Hum60:1-560
[34] Bornschein J, Rokkas T, Selgrad M,&Malfertheiner P (2009) Helicobacter pyloriand clinical aspects of gastric cancer. Helicobacter14Suppl1:41-45
[35] Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell136(2):215-233
[36] Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer.Nat Rev Genet10(10):704-714
[37] Tavazoie SF, et al.(2008) Endogenous human microRNAs that suppress breast cancermetastasis. Nature451(7175):147-152
[38] Meng F, et al.(2007) MicroRNA-21regulates expression of the PTEN tumorsuppressor gene in human hepatocellular cancer. Gastroenterology133(2):647-658
[39] Bandi N, et al.(2009) miR-15a and miR-16are implicated in cell cycle regulation in aRb-dependent manner and are frequently deleted or down-regulated in non-small celllung cancer. Cancer Res69(13):5553-5559
[40] Petrocca F, et al.(2008) E2F1-regulated microRNAs impair TGFbeta-dependentcell-cycle arrest and apoptosis in gastric cancer. Cancer Cell13(3):272-286
[41] Ueda T, et al.(2010) Relation between microRNA expression and progression andprognosis of gastric cancer: a microRNA expression analysis. Lancet Oncol11(2):136-146
[42] Liu T, Tang H, Lang Y, Liu M,&Li X (2009) MicroRNA-27a functions as anoncogene in gastric adenocarcinoma by targeting prohibitin. Cancer Lett273(2):233-242
[43] Ding L, et al.(2010) MiR-375frequently downregulated in gastric cancer inhibits cellproliferation by targeting JAK2. Cell Res20(7):784-793
[44] Zhao X, et al.(2012) MicroRNA-7functions as an anti-metastatic microRNA ingastric cancer by targeting insulin-like growth factor-1receptor. Oncogene
[45] Xiao B, et al.(2009) Induction of microRNA-155during Helicobacter pyloriinfection and its negative regulatory role in the inflammatory response. J Infect Dis200(6):916-925
[46] Zhang Z, et al.(2008) miR-21plays a pivotal role in gastric cancer pathogenesis andprogression. Lab Invest88(12):1358-1366
[47] Gao C, et al.(2010) Reduced microRNA-218expression is associated with highnuclear factor kappa B activation in gastric cancer. Cancer116(1):41-49
[48] Pallante P, et al.(2006) MicroRNA deregulation in human thyroid papillarycarcinomas. Endocr Relat Cancer13(2):497-508
[49] Wong QW, et al.(2010) MiR-222overexpression confers cell migratory advantagesin hepatocellular carcinoma through enhancing AKT signaling. Clin Cancer Res16(3):867-875
[50] Felicetti F, Errico MC, Segnalini P, Mattia G,&Care A (2008) MicroRNA-221and-222pathway controls melanoma progression. Expert Rev Anticancer Ther8(11):1759-1765
[51] Hutzinger R, Mrazek J, Vorwerk S,&Huttenhofer A (2010) NcRNA-microchipanalysis: a novel approach to identify differential expression of noncoding RNAs.RNA Biol7(5):586-595
[52] Kim YK, et al.(2009) Functional links between clustered microRNAs: suppression ofcell-cycle inhibitors by microRNA clusters in gastric cancer. Nucleic Acids Res37(5):1672-1681
[53] Zhang C, et al.(2009) Co-suppression of miR-221/222cluster suppresses humanglioma cell growth by targeting p27kip1in vitro and in vivo. Int J Oncol34(6):1653-1660
[54] Zhao JJ, et al.(2008) MicroRNA-221/222negatively regulates estrogen receptoralpha and is associated with tamoxifen resistance in breast cancer. J Biol Chem283(45):31079-31086
[55] Miller TE, et al.(2008) MicroRNA-221/222confers tamoxifen resistance in breastcancer by targeting p27Kip1. J Biol Chem283(44):29897-29903
[56] Terasawa K, Ichimura A, Sato F, Shimizu K,&Tsujimoto G (2009) Sustainedactivation of ERK1/2by NGF induces microRNA-221and222in PC12cells. FEBS J276(12):3269-3276
[57] Liu X, et al.(2009) MicroRNA-222regulates cell invasion by targeting matrixmetalloproteinase1(MMP1) and manganese superoxide dismutase2(SOD2) intongue squamous cell carcinoma cell lines. Cancer Genomics Proteomics6(3):131-139
[58] Oh J, et al.(2001) The membrane-anchored MMP inhibitor RECK is a key regulatorof extracellular matrix integrity and angiogenesis. Cell107(6):789-800
[59] Song SY, Son HJ, Nam E, Rhee JC,&Park C (2006) Expression ofreversion-inducing-cysteine-rich protein with Kazal motifs (RECK) as a prognosticindicator in gastric cancer. Eur J Cancer42(1):101-108
[60] Garofalo M, Quintavalle C, Romano G, Croce CM,&Condorelli G (2012)miR221/222in cancer: their role in tumor progression and response to therapy. CurrMol Med12(1):27-33
[61] Chun-Zhi Z, et al.(2010) MicroRNA-221and microRNA-222regulate gastriccarcinoma cell proliferation and radioresistance by targeting PTEN. BMC Cancer10:367
[62] le Sage C, et al.(2007) Regulation of the p27(Kip1) tumor suppressor by miR-221and miR-222promotes cancer cell proliferation. EMBO J26(15):3699-3708
[63] Visone R, et al.(2007) MicroRNAs (miR)-221and miR-222, both overexpressed inhuman thyroid papillary carcinomas, regulate p27Kip1protein levels and cell cycle.Endocr Relat Cancer14(3):791-798
[64] Fu X, et al.(2011) Clinical significance of miR-221and its inverse correlation withp27Kip(1) in hepatocellular carcinoma. Mol Biol Rep38(5):3029-3035
[65] Garofalo M, et al.(2008) MicroRNA signatures of TRAIL resistance in humannon-small cell lung cancer. Oncogene27(27):3845-3855
[66] Fornari F, et al.(2008) MiR-221controls CDKN1C/p57and CDKN1B/p27expression in human hepatocellular carcinoma. Oncogene27(43):5651-5661
[67] Zhang C, et al.(2010) PUMA is a novel target of miR-221/222in human epithelialcancers. Int J Oncol37(6):1621-1626
[68] Di Leva G, et al.(2010) MicroRNA cluster221-222and estrogen receptor alphainteractions in breast cancer. J Natl Cancer Inst102(10):706-721
[69] Garofalo M, et al.(2009) miR-221&222regulate TRAIL resistance and enhancetumorigenicity through PTEN and TIMP3downregulation. Cancer Cell16(6):498-509
[70] Pineau P, et al.(2010) miR-221overexpression contributes to liver tumorigenesis.Proc Natl Acad Sci U S A107(1):264-269
[71] Felli N, et al.(2005) MicroRNAs221and222inhibit normal erythropoiesis anderythroleukemic cell growth via kit receptor down-modulation. Proc Natl Acad Sci US A102(50):18081-18086
[72] Takahashi C, et al.(1998) Regulation of matrix metalloproteinase-9and inhibition oftumor invasion by the membrane-anchored glycoprotein RECK. Proc Natl Acad Sci US A95(22):13221-13226
[73] Clark JC, Thomas DM, Choong PF,&Dass CR (2007) RECK--a newly discoveredinhibitor of metastasis with prognostic significance in multiple forms of cancer.Cancer Metastasis Rev26(3-4):675-683
[74] Teng GG, et al.(2013) Let-7b Is Involved in the Inflammation and ImmuneResponses Associated with Helicobacter pylori Infection by Targeting Toll-LikeReceptor4. PLoS One8(2):e56709
[75] Zabaleta J (2012) MicroRNA: A Bridge from H. pylori Infection to Gastritis andGastric Cancer Development. Front Genet3:294
[76] Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell116(2):281-297
[77] Ambros V (2004) The functions of animal microRNAs. Nature431(7006):350-355
[78] Taganov KD, Boldin MP,&Baltimore D (2007) MicroRNAs and immunity: tinyplayers in a big field. Immunity26(2):133-137
[79] Xiao C&Rajewsky K (2009) MicroRNA control in the immune system: basicprinciples. Cell136(1):26-36
[80] Gottwein E&Cullen BR (2008) Viral and cellular microRNAs as determinants ofviral pathogenesis and immunity. Cell Host Microbe3(6):375-387
[81] Jopling CL, Yi M, Lancaster AM, Lemon SM,&Sarnow P (2005) Modulation ofhepatitis C virus RNA abundance by a liver-specific MicroRNA. Science309(5740):1577-1581
[82] Jopling CL (2008) Regulation of hepatitis C virus by microRNA-122. Biochem SocTrans36(Pt6):1220-1223
[83] Chen XM, Splinter PL, O'Hara SP,&LaRusso NF (2007) A cellular micro-RNA,let-7i, regulates Toll-like receptor4expression and contributes to cholangiocyteimmune responses against Cryptosporidium parvum infection. J Biol Chem282(39):28929-28938
[84] Liu Z, et al.(2010) Up-regulated microRNA-146a negatively modulate Helicobacterpylori-induced inflammatory response in human gastric epithelial cells. MicrobesInfect12(11):854-863
[85] Yoshida N, et al.(1993) Mechanisms involved in Helicobacter pylori-inducedinflammation. Gastroenterology105(5):1431-1440
[86] Mooney C, et al.(1991) Neutrophil activation by Helicobacter pylori. Gut32(8):853-857
[87] Takashima M, Furuta T, Hanai H, Sugimura H,&Kaneko E (2001) Effects ofHelicobacter pylori infection on gastric acid secretion and serum gastrin levels inMongolian gerbils. Gut48(6):765-773
[88] Perry MM, et al.(2008) Rapid changes in microRNA-146a expression negativelyregulate the IL-1beta-induced inflammatory response in human lung alveolarepithelial cells. J Immunol180(8):5689-5698
[89] Li J, et al.(2010) Altered microRNA expression profile with miR-146a upregulationin CD4+T cells from patients with rheumatoid arthritis. Arthritis Res Ther12(3):R81
[90] Brandt S, Kwok T, Hartig R, Konig W,&Backert S (2005) NF-kappaB activation andpotentiation of proinflammatory responses by the Helicobacter pylori CagA protein.Proc Natl Acad Sci U S A102(26):9300-9305
[91] Hou J, et al.(2009) MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFNproduction in macrophages by targeting TRAF6, IRAK1, and IRAK2. J Immunol183(3):2150-2158
[92] Dai R, et al.(2008) Suppression of LPS-induced Interferon-gamma and nitric oxide insplenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism ofimmune modulation. Blood112(12):4591-4597
[93] Nahid MA, Pauley KM, Satoh M,&Chan EK (2009) miR-146a is critical forendotoxin-induced tolerance: IMPLICATION IN INNATE IMMUNITY. J Biol Chem284(50):34590-34599
[94] Bhaumik D, et al.(2008) Expression of microRNA-146suppresses NF-kappaBactivity with reduction of metastatic potential in breast cancer cells. Oncogene27(42):5643-5647
[95] Fassi Fehri L, et al.(2010) Helicobacter pylori induces miR-155in T cells in acAMP-Foxp3-dependent manner. PLoS One5(3):e9500
[1] Taganov KD, Boldin MP, Baltimore D, et al. MicroRNAs and immunity: tinyplayers in a big field [J]. Immunity.2007,26(2):133-137.
[2] Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, Lim LP, et al. The widespreadimpact of mammalian microRNAs on mRNA repression and evolution. Science2005;310:1817–21.
[3] Zhou F, Ferguson J, Chang JT. Inter-and intra-combinatorial regulation bytranscription factors and microRNAs. BMC Genomics2007;8:396.
[4] Sylvestre Y, De Guire V, Querido E, Mukhopadhyay UK, Bourdeau V, Major F, et al.An E2F/miR-20a autoregulatory feedback loop. J Biol Chem2007;282:2135–43.
[5] Espel E. The role of the AU-rich elements of mRNAs in controlling translation. SeminCell Dev Biol2005;16:59–67.
[6] Bakheet T, Frevel M, Williams BR, Greer W, Khabar KS. ARED: human AU-richelement-containing mRNA database reveals an unexpectedly diverse functionalrepertoire of encoded proteins. Nucleic Acids Res2001;29:246–54.
[7] Zubiaga AM, Belasco JG, Greenberg ME. The nonamer UUAUUUAUU is the keyAU-rich sequence motif that mediates mRNA degradation. Mol CellBiol1995;4:2219–30.
[8] Cao H, Deterding LJ, Blackshear PJ. Phosphorylation site analysis of theanti-inflammatory and mRNA-destabilizing protein tristetraprolin. Expert RevProteomics2007;4:711–26.
[9] Peng SS, Chen CY, Xu N, Shyu AB. RNA stabilization by the AU-rich elementbinding protein, HuR, an ELAV protein. EMBO J1998;17:3461–70.
[10] Loflin P, Chen CY, Shyu AB. Unraveling a cytoplasmic role for hnRNP D in the invivo mRNA destabilization directed by the AU-rich element. Genes Dev1999;13:1884–97.
[11] Lai WS, Blackshear PJ. Interactions of CCCH zinc finger proteins with mRNA:tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur inthe absence of a poly [A] tail. J Biol Chem2001;276:23144–54.
[12] Moschos SA, Williams AE, Perry MM, Birrell MA, Belvisi MG, Lindsay MA.Expression profiling in vivo demonstrates rapid changes in lung microRNA levelsfollowing lipopolysaccharide-induced inflammation but not in the anti-inflammatoryaction of glucocorticoids. BMC Genomics2007;8:240.
[13] Tili E, Michaille J-J, Cimino A, et al. Modulation of miR-155and miR-125b levelsfollowing lipopolysaccharide/TNF-αstimulation and their possible roles in regulatingthe response to endotoxin shock. J Immunol,2007,179(8):5082～5089
[14] Cameron J E, Yin Q Y, Fewell C, et al. The Epstein-Barr Virus latent membraneprotein1(LMP1) induces cellular microRNA-146a, a modulator of lymphocytesignaling pathways. J Virol,2008,82(4):1946～1958.
[15] Perry MM, Moschos SA, Williams AE, et al. Rapid changes in microRNA-146aexpression negatively regulate the IL-1β induced inflammatory response in humanlung alveolar epithelial cells［J］. J Immunol,2008,180(8):5689-5698.
[16] Jing Q, Huang S, Guth S, et al. Involvement of microRNA in AU-richelement-mediated mRNA instability. Cell,2005,120(5):623～634.
[17] Vasudevan S, Tong Y C, Steitz J A. Switching from repression to activation:microRNAs can up-regulate translation. Science,2007,318(5858):1931～1934.
[1] Deretic, V.(2009). Multiple regulatory and effector roles of autophagy in immunity.Curr Opin Immunol21,53-62.
[2] Mizushima, N., Yoshimori, T. and Levine, B.(2010). Methods in mammalianautophagy research. Cell140,313-26.
[3] Levine, B., Mizushima, N. and Virgin, H.W.(2011). Autophagy in immunity andinflammation. Nature469,323-35.
[4] Yang, Z. and Klionsky, D.J.(2010). Mammalian autophagy: core molecular machineryand signaling regulation. Curr Opin Cell Biol22,124-31.
[5] Salgame, P.(2005). Host innate and Th1responses and the bacterial factors thatcontrol Mycobacterium tuberculosis infection. Curr Opin Immunol17,374-80.
[6] Gutierrez, M.G., Master, S.S., Singh, S.B., Taylor, G.A., Colombo, M.I. and Deretic, V.(2004). Autophagy is a defense mechanism inhibiting BCG and Mycobacteriumtuberculosis survival in infected macrophages. Cell119,753-66.
[7] Singh, S.B., Davis, A.S., Taylor, G.A. and Deretic, V.(2006). Human IRGM inducesautophagy to eliminate intracellular mycobacteria. Science313,1438-41.
[8] Harris, J., De Haro, S.A., Master, S.S., Keane, J., Roberts, E.A., Delgado, M. andDeretic, V.(2007). T helper2cytokines inhibit autophagic control of intracellularMycobacterium tuberculosis. Immunity27,505-17.
[9] Andrade, R.M., Wessendarp, M., Gubbels, M.J., Striepen, B. and Subauste, C.S.(2006). CD40induces macrophage anti-Toxoplasma gondii activity by triggeringautophagy-dependent fusion of pathogen-containing vacuoles and lysosomes. J ClinInvest116,2366-77.
[10] Baregamian, N., Song, J., Bailey, C.E., Papaconstantinou, J., Evers, B.M. and Chung,D.H.(2009). Tumor necrosis factor-alpha and apoptosis signal-regulating kinase1control reactive oxygen species release, mitochondrial autophagy, and c-JunN-terminal kinase/p38phosphorylation during necrotizing enterocolitis. Oxid MedCell Longev2,297-306.
[11] Harris, J. and Keane, J.(2010). How tumour necrosis factor blockers interfere withtuberculosis immunity. Clin Exp Immunol161,1-9.
[12] Jia, G., Cheng, G., Gangahar, D.M. and Agrawal, D.K.(2006). Insulin-like growthfactor-1and TNF-alpha regulate autophagy through c-jun N-terminal kinase and Aktpathways in human atherosclerotic vascular smooth cells. Immunol Cell Biol84,448-54.
[13] Jia, L., Dourmashkin, R.R., Allen, P.D., Gray, A.B., Newland, A.C. and Kelsey, S.M.(1997). Inhibition of autophagy abrogates tumour necrosis factor alpha inducedapoptosis in human T-lymphoblastic leukaemic cells. Br J Haematol98,673-85.
[14] Keller, C.W., Fokken, C., Turville, S.G., Lunemann, A., Schmidt, J., Munz, C. andLunemann, J.D.(2011). TNF-alpha induces macroautophagy and regulates MHC classII expression in human skeletal muscle cells. J Biol Chem286,3970-80.
[15] Ling, Y.M., Shaw, M.H., Ayala, C., Coppens, I., Taylor, G.A., Ferguson, D.J. and Yap,G.S.(2006). Vacuolar and plasma membrane stripping and autophagic elimination ofToxoplasma gondii in primed effector macrophages. J Exp Med203,2063-71.
[16] Sivaprasad, U. and Basu, A.(2008). Inhibition of ERK attenuates autophagy andpotentiates tumour necrosis factor-alpha-induced cell death in MCF-7cells. J Cell MolMed12,1265-71.
[17] Djavaheri-Mergny, M., Amelotti, M., Mathieu, J., Besancon, F., Bauvy, C., Souquere,S., Pierron, G. and Codogno, P.(2006). NF-kappaB activation represses tumor necrosisfactor-alpha-induced autophagy. J Biol Chem281,30373-82.
[18] Arico, S., Petiot, A., Bauvy, C., Dubbelhuis, P.F., Meijer, A.J., Codogno, P. andOgier-Denis, E.(2001). The tumor suppressor PTEN positively regulatesmacroautophagy by inhibiting the phosphatidylinositol3-kinase/protein kinase Bpathway. J Biol Chem276,35243-6.
[19] Van Grol, J., Subauste, C., Andrade, R.M., Fujinaga, K., Nelson, J. and Subauste, C.S.(2010). HIV-1inhibits autophagy in bystander macrophage/monocytic cells throughSrc-Akt and STAT3. PLoS One5, e11733.
[20] Ni Cheallaigh, C., Keane, J., Lavelle, E.C., Hope, J.C. and Harris, J.(2011).Autophagy in the immune response to tuberculosis: clinical perspectives. Clin ExpImmunol164,291-300.
[21] Kiyono, K., Suzuki, H.I., Matsuyama, H., Morishita, Y., Komuro, A., Kano, M.R.,Sugimoto, K. and Miyazono, K.(2009). Autophagy is activated by TGF-beta andpotentiates TGF-beta-mediated growth inhibition in human hepatocellular carcinomacells. Cancer Res69,8844-52.
[22] Roca, H., Varsos, Z.S., Sud, S., Craig, M.J., Ying, C. and Pienta, K.J.(2009). CCL2and interleukin-6promote survival of human CD11b+peripheral blood mononuclearcells and induce M2-type macrophage polarization. J Biol Chem284,34342-54.
[23] Harris, J. et al.(2011). Autophagy controls IL-1beta secretion by targetingpro-IL-1beta for degradation. J Biol Chem286,9587-97.
[24] Fujishima, Y. et al.(2011). Autophagy in the intestinal epithelium reducesendotoxin-induced inflammatory responses by inhibiting NF-kappaB activation. ArchBiochem Biophys506,223-35.
[25] Li, Y.Y. et al.(2011). Autophagy is required for toll-like receptor-mediatedinterleukin-8production in intestinal epithelial cells. Int J Mol Med27,337-44.
[26] Lee, H.K., Lund, J.M., Ramanathan, B., Mizushima, N. and Iwasaki, A.(2007).Autophagy-dependent viral recognition by plasmacytoid dendritic cells. Science315,1398-401.
[27] Tal, M.C., Sasai, M., Lee, H.K., Yordy, B., Shadel, G.S. and Iwasaki, A.(2009).Absence of autophagy results in reactive oxygen species-dependent amplification ofRLR signaling. Proc Natl Acad Sci U S A106,2770-5.
[28] Saitoh, T. et al.(2008). Loss of the autophagy protein Atg16L1enhancesendotoxin-induced IL-1beta production. Nature456,264-8.