1. The Role And Mechanism Of FoxMi In H.Pylori-related Gastric Cancer2. The Role And Mechanism Of MiR-146a In Morphine-induced Apoptosis

BackgroundGastric cancer is the fourth prevalent cancer and the second cause of cancer-related death worldwide, especially in developing countries. Gastric carcinogenesis is a complex multi-step and multi-factorial event. It has been extensively recognized that H.pylori infection is involved in the early stage of gastric cancer pathogenesis through induction of chronic gastritis, and that interaction of host-pathogen contributes to this carcinogenesis. H.pylori has been classified by the World Health Organization as a class1carcinogen in1994. It is well accepted that H.pylori and its key virulence factor CagA may be an initiating factor in the development process of gastric cancer, from chronic gastritis, peptic ulcer, intestinal metaplasia, atypical hyperplasia to gastric cancer. To date, however, the underlying molecular mechanism of carcinogensis relatived with H.pylori remains to be elucidated.It has been proved that infection and inflammation was involved in carcinogenesis. Chronic inflammation elicited by H.pylori has been confirmed as a critical risk in the progression of gastric cancer, and this chronic inflammation can persist for decades and result in the nonresolving inflammation, a major driver to gastric cancer. Fork head Box M1(FoxM1), a member of the Fox transcription factor family, is a key positive cell-cycleregulator in cell proliferation. Aberrant expressionof FoxM1is involved in several tumor types, including hepatocellular carcinoma, basal cell carcinoma, breast cancer, lung cancer, prostate cancer, glioblastomas and GC, which implies an oncogene role in carcinogenesis. We previously reported that FoxM1is upregulated in GC, and its inhibition leads to cellular senescence, but therelevance of H.pylori infection and FoxMl expression associated with the pathogenesis of GC remains undefined. MicroRNAs (miRNAs), a family of small noncoding RNAs, are important negative regulators of post-transcriptional gene expression bydirectly targeting the3’untranslated regions (3’UTRs) of target mRNAs, eventually promoting the degradation or translation suppression of target mRNAs. MiRNAs are frequently deregulated in many types of human cancers and play critical roles in tumorigenesis, serving as tumor suppressors or oncogenes. However, the role and mechanisms of miRNAs in H.pylori-related gastric carcinogenesis is still unknown.AimWe previously reported that FoxM1is upregulated in GC, and its inhibition leads to cellular senescence, but therelevance of H.pylori infection and FoxM1expression associated with the pathogenesis of GC remains undefined. In this study, we attempted to explore the effect of H.pylori infection on FoxM1expression and possible mechanisms.Methods and Results1. The analysis of FoxM1expression in various gastric tissues. The expression of FoxM1was analyzed by Real-time PCR and IHC in various gastric specimens at different disease stages, including normal, superficial gastritis (SG), atrophic gastritis associated with intestinal metaplasia (AG/IM) and primary GC tissues. FoxM1expression was gradually increased in human gastric species from inflammation to cancer.2. The effect of FoxM1on H.pylori-induced proliferation of gastric cancer cell in vitro. The gastric epithelial-derived cell lines infected with H.pylori showed up-regulation of FoxM1in the manner of time and concentration dependent. CagA, the key virulence factor of H.Pylori, showed the ability of increasing FoxMl expression.H.pylori and CagA inhibited expression of P27Kip1and subsequent promoted cells proliferation through up-regulating of FoxMl.3. Prediction and identification of FoxMl as a direct target of miR-370, analysis the Correlation between miR-370and H. pylori-induced cell proliferation in gastric cancer cells. The expression of miR-370was declined in human species of gastritis and gastric cancer by Real-time PCR. We identified that FoxMl was directly down-regulated by hsa-miR-370in the gastric cell lines by western blot and Luciferase assay. H.pylori and CagA inhibited miR-370which led to overexpression of FoxM1and cell proliferation.4. miR-370-FoxMl is involved in H.pylori-induced gastritis in vivo. The validation of mouse model of gastritis induced by H.pylori infection was confirmed by hematoxylin and eosin staining. After administration of H.pylori for8months,90%(18/20) of mice were validated as gastritis models in the H.pylori infection group as compared with13%(2/15) in the control group. FoxM1protein level was greater in the model than the control on IHC assay. In addition, the mRNA level of mmu-miR-370was reduced and that of FoxMl increased in mucosal epithelial samples of H.pylori-induced gastritis. The results in vivo revealed the involvement of FoxMl and miR-370inH.pylori-induced gastritis.Conclusions1. FoxM1expression was gradually increased in human gastric species from inflammation to cancer. Overexpression of FoxM1is an early event in H.pylori-relted gastric carcinogenesis.2. H.pylori and its key virulence factor CagA promotes FoxM1expression in gastric epithelial cell lines. 3. H.pylori and CagA were involved in cell proliferation by suppressing p27kipl expression mediated by FoxMl.4. FoxMl is a direct target of hsa-miR-370and miR-370-FoxM1pathway mediates H. pylori-induced cell proliferation.5. FoxM1is involved in H. pylori-induced gastritis in vivo. BackgroudMorphine is one of the most potent pain reliever for centuries, but also a potent inducer of dependence.The uncontrolled use of opioids has been demonstrated to result in significant deterioration of the immune system.The mechanisms, however, remian to be identified. MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that suppress protein expression mainly by binding to the3’ untranlated region (UTR) of their target messenger RNAs. Here, we report that morphine can reduce the expression of microRNA-146a (miR-146a). MiR-146a blocks morphine-induced cell apoptosis by targeting the Fas/FADD pathway. Enforced expression of miR-146a diminishes morphine-induced cell apoptosis both in vitro and in vivo. Most interestingly, administration of miR-146a mimic in mice markedly attenuates morphine physical dependence.AimOur discovery of modulation of miR-146a by opioids will enhance the understanding of the interactions between the immune and nervous systems. Current and further determinations of the function and mechanism of this novel signal will lead to our understanding of miRNA and apoptosis during the development of opioid dependence, providing potential molecular targets of novel therapeutics to treat opioid dependence and apoptosis. Methods and Results1. The effect of morphine administration on the expression of miR-146a.To identify which miRNA was regulated by morphine, macrophage cell line RAW264.7andmurine peritoneal macrophages were treated with morphine and the expression of different miRNAs was determined by real-time quantitative RT-PCR (qRT-PCR). We found that morphine markedly decreased the levels of miR-146a. To determine the in vivo effect of morphine on miR-146a regulation, wild type (WT) mice were administered with morphine. Consistent with the results in vitro experiments, morphine reduced the expression of miR-146a in the spleen and liver.2. Prediction and veryfiction of Fas and Fadd are direct targets of miR-146a.We then applied the prediction program miRanda21to identify new possible miR-146targets. Cell deadth receptor Fas and Fas-associated death domain (FADD) have putative miR-146a binding elements in their3’-UTRs.Luciferase reporter assay was performed and the result suggested that Fas and FADD are direct targets of miR-146a. Consistent with that, miR-146a diminished the expression of Fas and FADD in both mRNAand protein levels.3. The role and mechsnism of miR-146a in morphine-induced apoptosis in vivo and in vitro.To assess the potential roles of miRNA-146a in morphine-induced apoptosis, we transfected RAW264.7cells with miR-146a mimic and identified apoptotic cells by the terminal deoxynucleotidy transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The RAW264.7cells forced miR-146a expressionand exposed to morphine led to asignificantlower number of apoptotic cells than the cells transfected with control mimic and morphine treatment. Then miRNA-146a mimic was administered in WT mice during chronic morphine administration, tissuesfrom the mice injected with both miR-146a mimic and morphine showed a dramatic decrease in the percentage of apoptotic cells. Taken together, these results suggest that miR-146a contributes to an anti-apoptotic effect and miR-146a inhibits apoptosis through the suppression of morphine-induced pro-apoptotic proteins. 4. Analysis the role of miR-146a on the main symptoms of morphine withdrawal.Morphine dependence was evaluated by assessing withdrawal responses following administration of the opioid receptor antagonist naloxone. Notably, in administered mice with miR-146a mimic the main behavioral signs of morphine withdrawal were markedly attenuated, while the mice injected with control mimic still exhibited classic behavioral signs of withdrawal after administration of naloxone, suggesting that enforced expression of miR-146a in mice does not become dependent on morphine.Conclusion1. Morphine decreases the expression of miR-146a.2. Fas and Fadd are direct targets of miR-146a.3. miR-146a attenuates morphine-induced apoptosis.4. miR-146a blocks the main aymptoms of morphine withdrawal.