Study On The Mechanism Of Helicobacter Pylori CagA As A Negative Regulator Of Autophagy And Increased MiR-222/146a In Gastric Diseases

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 inflammatoryresponseWe 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 RECKRT-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 cellsWe 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.