Mechanism For Helicobacter Pylori Lipopolysaccharide-induced MiR-375and MiR-106b Inhibition In Gastric Cancer

Gastric cancer remains the second leading cause of cancer-related deaths in theworldwide, and a high incidence of gastric cancer has reported in China. Agram-negative Spiral bacterium, Helicobacter pylori (H. pylori) is estimated topresent in the stomachs of at least half of the human population, and also up to54.76%in Chinese pupation. The WHO stated in1994that H. pylori as a group1carcinogen. Ongoing research of gastric cancer suggests that H. pylori may play animportant role in the development of gastric cancer. However, while this associationis becoming increasingly recognized, the mechanisms involved remain unknown.MicroRNAs (miRNAs) are small non-coding RNA molecules approximately18-25nucleotides (nts) in length that serve as critical transcriptional regulators. For example,depending on the extent of complementarity between the3’ untranslated region (3’UTR) of a miRNA and its target gene, miRNAs can regulate mRNA degradation, ortranslational repression, thereby affecting gene expression.52.5%of miRNA genesare located in cancer-associated genomic regions or in fragile sites. Correspondingly,numerous studies have demonstrated that miRNAs play a role in the progression,diagnosis and treatment of human malignancies.Recent study has shown that miR-375, one of30miRNAs expressed at very lowlevels in gastric mucosa infected with H. pylori compared with normal gastric mucosa,closely correlates with the activity, chronic inflammation, and colonization densitythat characterize an H. pylori infection. However, down-regulation of miR-375ingastric cancer has also been shown to act as a tumor suppressor. In combination, thesedata suggest that miR-375plays a role in an infection by H. pylori and thedevelopment of gastric cancer. JAK2, identified as a direct target gene of miR-375, is major regulator of JAK/STAT3signal pathway that associated with inflammation andtumor. Using computational targeted gene predictions, JAK1and STAT3, which alsoinvolved in JAK/STAT3signal pathway, are shown potential targets sites formiR-106b. However, H. pylori infection inhibited miR-106b expression in gastricmucosa. Therefore, we hypothesized that the H. pylori infection down-regulatesmiR-375in gastric cancer to promote JAK2expression, on the other hand, elevatinglevels of JAK2and STAT3by inhibition of miR-106b, ultimately leading to triggerthe activation of JAK/STAT3signal pathway in gastric cancer.In this study, we used Lipopolysaccharide(LPS), a virulence factor of H. pylori, tostudy the mechanistic basis of H. pylori infection induce the dysregulation of miRNAin gastric cancer. We demonstrated that H. pylori-LPS regulates the expression ofmiR-375and miR-106b in gastric cancer to activate JAK/STAT3signal pathway andoffer a new sight of the role of H. pylori infection in the aggravation of themiRNA-induced inflammation and development of gastric cancer. Moreover, the H.pylori infection-induced miRNAs expression may be a novel target for diagnosis,treatment and prognosis of gastric cancer in the future.Methods1. Studies on the expression of miR-375and miR-106b in gastric cancer cells with theH. pylori-LPS stimulation.Following treatment of AGS and MKN45gastric cancer cells with H. pylori-LPS1μg/mL for12h, miR-375and miR-106b expression was detected using TaqManmiRNA assays. In experiments using TLR4inhibitors, AGS and MKN45cells inserum-free medium were pretreated for6h with40μg/mL CLi095, then cells treatedwith H. pylori-LPS1μg/mL for another12h. H. pylori-infected gastric mucosatissues and gastric cancer cells with H. pylori-LPS stimulation for12h were alsoanalyzed for Dicer expression by western blotting and real time PCR.2. Studies on the mechanism of H. pylori-LPS induced miR-375expression in gastriccancer cells.Following treatment of AGS and MKN45gastric cancer cells with H. pylori-LPS for12h, Sp1, MDM2and p63expression was detected using by western blotting and real time PCR. Interactions between miR-375and Sp1and MDM2were analyzed bywestern blotting, real time PCR, dual luciferase assays, and immunofluorescence.Overexpression and knockdown assays of MDM2were used to investigateinteractions between MDM2and Dicer, as well as p63.3. Studies on the mechanism of H. pylori-LPS induced miR-375and miR-106bexpression to active JAK/STAT3signal pathway.Following treatment of AGS and MKN45gastric cancer cells with H. pylori-LPS for12h, MCM7and JAK2expression were detected using by western blotting or realtime PCR. Overexpression of MDM2was used to investigate the expression ofmiR-106b. Interactions between miR-106b and JAK1and STAT3were analyzed bywestern blotting, real time PCR, dual luciferase assays. Following treatment of AGSand MKN45gastric cancer cells with H. pylori-LPS at various time points, Westernblotting was performed to detect the phosphorylation levels of JAK1, JAK2andSTAT3.Resultes1. Studies on the expression of miR-375and miR-106b in gastric cancer cells with theH. pylori-LPS stimulation.AGS and MKN45cells were treated with1μg/mL H. pylori-LPS for12h, decreasein expression of miR-375and miR-106b were observed compared with untreatedcells. Subsequently, AGS and MKN45cells were preincubated with Cli095, aTLR-4-specific inhibitor. Pretreatment with Cli095restored expression of miR-375and miR-106b following treatment with H. pylori-LPS compared with control cells (P<0.01). Levels of Dicer in10H. pylori-infected gastric tissue samples and10normaltissue samples were analyzed. In the western blots performed, levels of Dicer in H.pylori-infected tissue samples were lower than the Dicer levels detected in normaltissue samples (P <0.05). Similarly, RNA levels of Dicer were lower in8H.pylori-infected gastric tissue samples compared with8normal tissue samples thatwere analyzed using real time PCR (P <0.01). In AGS and MKN45cells followingtreatment with H. pylori-LPS for12h, both protein and mRNA levels of Dicer wasfound to decrease in both cell lines compared with that in control cells. 2、Studies on the mechanism of H. pylori-LPS induced miR-375expression in gastriccancer cells.2.1The role of Sp1and MDM2on the mechanism of H. pylori-LPS induced miR-375expressionBoth protein and mRNA levels of Sp1and MDM2were found to increase comparedwith untreated cells after stimulation with H. pylori-LPS for12h. With miR-375inhibitors transiently transfected into AGS and MKN45cells, a significant increase inSp1mRNA was detected by Real time PCR in both cell lines compared with that inuntreated cells (P <0.05). In contrast, a decrease in levels of Sp1mRNA wasobserved when AGS and MKN45cells were transfected with miR-375mimics.Similar results were obtained in western blotting assays of AGS and MKN45cellstransfected with miR-375inhibitors versus miR-375mimics followed by treatmentwith H. pylori-LPS (P <0.01in each case). AGS and MKN45cells transfected withpGL-MDM2-wt reporter plasmids, then treated with H. pylori-LPS (10μg/mL),exhibited enhanced luciferase activity compared with control cells (P <0.01). Atransient co-transfection of miR-375inhibitors and pGL-MDM2-wt reporter plasmidsinto MKN45cells resulted in an increase in luciferase activity for MDM2comparedwith control cells (P <0.05). In AGS and MKN45cells transfected with miR-375inhibitors, then treated with H. pylori-LPS, expression of MDM2significantlyincreased compared to untransfected cells.2.2The interaction of MDM2and Dicer on the mechanism of H. pylori-LPS inducedmiR-375expressionTransient expression assays were conducted in gastric cancer cells usingpcDNA3.1-MDM2plasmids. Expression of Dicer at both protein and mRNA levels,respectively, significantly decreased compared with untreated cells. Moreover,expression of miR-375in transfected cells was also inhibited. In contrast, proteinlevels of Dicer significantly increased in gastric cancer cells transfected with aMDM2-siRNA compared with untransfected cells. In AGS and MKN45cells treatedwith H. pylori-LPS for12h, total expression levels of p63were found to increase inboth AGS and MKN45cells compared with uninfected cells. In addition, total expression of p63significantly decreased in AGS and MKN45cells transfected withpcDNA3.1-MDM2plasmids.3. Studies on the mechanism of H. pylori-LPS induced miR-375and miR-106bexpression to active JAK/STAT3signal pathway.In AGS and MKN45cells treated with H. pylori-LPS for12h, the protein and mRNAlevels of MCM7were observed unchanged in cells compared with that in untreatedcells. The expression miR-106b was significantly down-regulated in AGS andMKN45cells transfected with pcDNA3.1-MDM2plasmids. Overexpression ofmiR-106b decreased JAK1and STAT3expression in AGS and MKN45cells at bothprotein and mRNA levels. A transient co-transfection of miR-106b mimics andpGL-JAK1-wt reporter plasmids into AGS and MKN45cells resulted in a decrease inluciferase activity for JAK1compared with control cells (P <0.01). However, thisdecrease was absent when AGS and MKN45cells were transfected with apGL-JAK1-mut reporter. Similar results were obtained in AGS and MKN45cellstransfected with miR-106b mimics and pGL-STAT3-wt or pGL-STAT3-mut reporterplasmids. In AGS and MKN45cells treated with1μg/mL H. pylori-LPS at varioustime points (e.g.0,1,10,30,60min), the phosphorylation levels of JAK1, JAK2andSTAT3protein was observed to increase in both cells lines compared with that inuninfected cells respectively. Using western blotting, treatment with H. pylori-LPSresulted in significant increases in levels of total JAK2protein, that was detected inAGS and MKN45cells for12h.Conclusions1．H. pylori-LPS induces miR-375and miR-106b down-regulation in gastric cancercells and Dicer is essential for regulating process of H. pylori-LPS-indecued miR-375and miR-106b expression.2．H. pylori-LPS decrease miR-375expression in gastric cancer cells, therebyactivating expression of MDM2via Sp1and inhibiting Dicer. Cumulatively, thisseries of events led to a reduction in levels of miR-375, and the induction of apositive feedback loop via a Sp1/MDM2/Dicer pathway in gastric cancer cells.3. JAK1and STAT3are direct targets of miR-106b in gastric cancer cells. Together with JAK2regulated by miR-375, H. pylori-LPS activate JAK/STAT3signalpathway via inhibition of miR-375and miR-106b.4. MDM2gene plays an important role in H. pylori-LPS-induced miRNA expressionin gastric cancer cells.