Regulation Mechanism Of Helicobacter Pylori-promoted Clock Gene BMAL1 Expression To Mediate Gastric Inflammation And Malignant Transformation

Background:Molecules,cells,the body,even to the group and the other levels,there are obvious clock periods in the life process.The important role of the circadian clock is to make the organism adapt to the environment better,and maintain the stability of the organism’s physiological function.Circadian rhythm can take 24 hours as a cycle to control a series of physiological processes,including sleep-wake cycles,metabolism and hormone secretion.The circadian rhythm system contains at least 12 genes,which could form multiple feedback loops and regulate approximately 10%of all the genes in the body.These genes are involved in diverse biological processes,including cell proliferation,apoptosis,migration and invasion.When this homeostasis is disrupted,organ-specific diseases such as obesity,diabetes,autoimmunity,and cancer will occur.Gastric cancer(GC)is a major disease affecting human health.The World Health Organization/International Agency for Research on Cancer(WHO/IARC)has identified Helicobacter pylori(H.pylori)as a type I carcinogen and it is a risk factor for GC.H.pylori infection mediates malignant transformation and undergoes an evolution of chronic superficial gastritis-chronic atrophic gastritis with intestinal metaplasia-dysplasia-GC.Gastritis paves the way for malignant transformation of gastric mucosa.The underlying mechanisms involved are complex and have not yet been fully elucidated.Gastrointestinal cells can independently regulate circadian rhythm molecules,making cells have circadian rhythm,suggesting that local factors may affect the expression of circadian clock genes in a manner that directly acts on specific cells.Interestingly,H.pylori showed effects on circadian gastric acidity which implied the potential relationship between circadian rhythm and H.pylori induced disorders.Several other studies uncovered the connection between circadian rhythm and inflammatory diseases.Therefore,we assumed that circadian rhythm may be crucial for the development of inflammatory disease induced by H.pylori.At the same time,it is important to explore the regulation mechanism through which rhythm genes contributed to malignant transformation of gastric epithelial cells,and understand the law of the occurrence and development of GC.Our previous screening found that the expression of clock gene BMAL1 was significantly up-regulated under conditions of H.pylori infection and in the process of malignant transformation of gastric epithelial cells.BMAL1 acts as a transcription factor at the core regulatory position in the rhythm molecular feedback loop.Studies have shown that when BMAL1 is abnormally expressed in monocytes,endothelial cells,epithelial cells and mesenchymal cells,it could affect a series of inflammatory reactions including pneumonia,arthritis and thrombosis,through regulating cytokines,chemokines,transcription factors,glucocorticoid receptors and metabolic related molecules.In addition,BMAL1 is abnormally expressed in hepatocellular carcinoma,head and neck squamous cell carcinoma,leukemia,nasopharyngeal carcinoma and the like.Therefore,this study is to investigate the role of the clock gene BMAL1 in the H.pylori induced gastritis,and the regulation mechanism of abnormal expression of BMAL1 promoting GC.This study aims to explain the importance of circadian genes in the development of gastric diseases by illustrating the role of circadian rhythm genes in different stages of gastritis progressing to GC.Aims1.Explore the relationship between clock gene BMAL1 and H.pylori infection-induced inflammatory cytokine expression and the progression of gastritis,and the underlying molecular mechanisms.2.Explore the role of clock gene BMAL1 in the progressive deterioration process from chronic gastritis to GC,and illustrate the underlying mechanisms.Methods and Results1.H.pylori induces LIN28A expression,which is an RNA binding protein,and LIN28A binds to the promoter region of circadian rhythm gene BMAL1 and directly activates its transcription under H.pylori infection,breaking the circadian rhythm of BMAL1.BMAL1 in turn functions as a transcription factor and enhances the expression of proinflammatory cytokine TNF-a,thereby promoting inflammation.(1)The relative expression of traditional circadian genes in public microarray data from GEO database were analyzed and Bmall expression was significantly increased during H.pylori infection in the mouse-derived cell lines.Immunohistochemistry(IHC)staining showed that in patient tissues from superficial gastritis(SG),atrophic gastritis(AG)to Intestinal metaplasia(IM)with or without H.pylori infection,the expression of BMAL1 in H.pylori infected patients was significantly higher than that in uninfected patients.Correspondingly,RT-PCR analysis in another cohort of specimens showed BMAL1 was also higher in H.pylori-positive patients than that in H.pylori-negative samples.In the in vitro experiment,gastric cell lines AGS and BGC-823 were incubated with two H.pylori strains(H.p 11637 and H.p 26695),and BMAL1 expression was increased in cells with H.pylori infection according to RT-PCR and Western blot analysis,and the up-regulation was time and concentration dependent.In gastric epithelial cells with reset circadian time,RT-PCR showed that H.pylori infection disrupted the rhythm of BMAL1 expression.(2)Targetscan website shows BMAL1 contains a binding site of let-7a-3p in the 3’UTR region.RT-PCR and Western blot showed the inhibition of LIN28A and BMAL1 by let-7a-3p.However,the results of the dual luciferase assay showed the inability of let-7a-3p to activate the transcription of BMAL1 through the binding site of its 3’UTR.Based on the above results,we hypothesized the regulation of BMAL1 by let-7a-3p through LIN28A.After overexpression or depletion of LIN28A,RT-PCR and Western blot showed corresponding up-or down-regulation of BMAL1.Chromatin immunoprecipitation assay(ChIP)showed the direct binding of LIN28A to BMAL1’s promoter.The dual luciferase assay was used to confirm the effect of LIN28A on the transcriptional activation of BMAL1.(3)RT-PCR and Western blot analysis showed that H.pylori promoted LIN28A expression in a time and concentration dependent way.Comparing "LIN28A si+Hp26695" with "NC+Hp26695" group,RT-PCR and Western blot also showed that induction of BMAL1 by H.pylori was alleviated upon LIN28A suppression.In addition,IHC staining demonstrated the expression of LIN28A was higher in H.pylori-positive patients than that in H.pylori-negative patients,and the differences were reflected by the relative values of IHC optical density.(4)H.pylori significantly activated Tnf expression in the mouse-derived cell lines upon analysis of the GEO database.In RT-PCR analysis,decline of TNF-a was found to be the most pronounced among the selected factors by BMAL1 depletion in human cell lines.Comparing "BMAL1 si+Hp26695" with "NC+Hp26695"group,induction of TNF-a RNA expression by H.pylori was relived upon BMAL1 inhibition.Accordingly,overexpression of BMAL1 showed the opposite effect.Above two results were all verified by RT-PCR experiments.Enzyme-Linked ImmunoSorbent Assay(ELISA)showed that the change of TNF-a secretion was align with that of its RNA.Since BMAL1 functions as circadian rhythm gene,TNF-α is supposed to be rhythmic as its target.RT-PCR and ELISA experiments showed that the rhythm of TNF-a in rhythm-synchronized cells was destroyed by infection of H.pylori.Correspondingly,when BMAL1 was inhibited,the rhythm change of TNF-a caused by H.pylori was restored.(5)Based on the distribution of E-box elements in the TNF-α promoter region,we used KOD-Plus-Mutagenesis Kit to knock them out separately and used double luciferase assay to detect the transcriptional activation of BMAL1.It was found that when the E-box elements of the K05 group were removed,the transcriptional activation of TNF-a by BMAL1 was not changed even though BMAL1 was inhibited.Since there are two E-box elements in the K05 group,these two sites were mutated with KOD-Plus-Mutagenesis Kit and tested with double luciferase assay separately.Both sites were found critical for BMAL1 activation.Furthermore we confirmed the direct binding of BMAL1 to the above two sites in TNF-α promoter by ChIP.(6)A mouse model was designed and generated.Briefly there are 4 groups of mice,of which 2 groups were subjected to 12 hours of light-12 hours of dark(LD)cycle plus intragastric administration(IG)with PBS or H.pylori strain SSI at 12 pm respectively.The other 2 groups were raised under constant darkness(DD)plus intragastric administration(IG)with PBS or H.pylori strain SSI at 12 pm respectively.RT-PCR and IHC showed that H.pylori infection up-regulated Bmall by two times.We used May-Grunwald-Giemsa kit to stain H.pylori,and found that the number of mice colonized by H.pylori was significantly increased in the "DD+SSI" group with the most abnormal expression of the clock gene Bmall.This indicated that the abnormal expression of rhythm molecules could promote the colonization of H.pylori.Inflammation indexes were scored according to H.pylori infection rate as well as the cell type,depth and location of inflammatory cell infiltration.Comparing with "LD+SSI" group,"DD+SSI" mice showed higher inflammation indexes,indicating the involvement of the rhythm molecules’ abnormal expression in the process of inflammation induced by H.pylori.The infiltration of inflammatory cells was observed after hematoxylin-eosin(HE)staining,and we found the "DD+SSI" group had the most cases and the highest proportion of mice with inflammatory cells infiltrating both the mucosal layer and the submucosa according to inflammatory response position.Similarly,RT-PCR analysis showed that,comparing with "LD+SSI" group,Tnf expression from mouse epithelial cells stimulated by H.pylori was more significantly increased in the "DD+SSI" group.2.IL-1β/IL-1R1-NF-κB induced BMAL1 activated IL-1R1 expression,forming the mutual regulation and positive feedback circuit between BMAL1 and IL-1β/IL-1R1 to promote overexpression of BMAL1 in gastric cancer.BMAL1 directly targeted cyclin D1 to promote gastric cancer cell abnormal proliferation.(1)By analyzing the GEO database,the rhythm gene BMAL1 was found to increase in intestinal metaplasia tissues progressing to gastric cancer(GC).We used RT-PCR to examine BMAL1,CLOCK,CRY1 and CRY2 in atrophic gastritis(AG)and GC,and found that only BMAL1 was significantly elevated in GC tissues.Immunehistochemistry(IHC)staining showed upregulation of BMAL1 during different stages of cancer progression,and the upregulation were reflected by the relative values of IHC optical density.We selected another cohort of specimens of GC patients with cancer and adjacent normal tissues for evaluation.RT-PCR and Western blot showed that BMAL1 was significantly elevated in cancerous tissues.(2)Cloning formation experiments and 5-ethynyl-2’-deoxyuridine(EdU)staining assay showed that BMAL1 could promote GC cell proliferation in vitro.Mouse model with subcutaneously injected GC cells into nude mice showed that BMAL1 could promote tumorigenesis in vivo.(3)RT-PCR and Western blot showed that BMAL1 could promote cyclin D1 expression.Cloning formation experiments and 5-ethynyl-2’-deoxyuridine(EdU)staining assay showed that cyclin D1 depletion obviously attenuated the increase of proliferative cells caused by BMAL1 overexpression.The dual luciferase assay and chromatin immunoprecipitation(ChIP)assay showed that BMAL1 combined with the cyclin D1 promoter and directly transcriptionally activated cyclin D1.(4)RT-PCR and Western blot showed that exogenous IL-1β stimulation upregulated BMAL1.Western blot showed that NF-κB signaling pathway inhibitor inhibited the upregulation of BMAL1 induced by IL-1β.RT-PCR and Western blot showed that NF-κB signaling pathway could mediate the upregulation of BMAL1 induced by IL-1β.The ChIP assay and dual luciferase assay showed that P65 combined with the BMAL1 promoter and directly transcriptionally activated BMAL1.(5)Public microarray data from GEO database was analyzed and IL1R1 was found to increase in intestinal metaplasia tissues progressing to GC.In the correlation analysis of data,IL-1R1 showed the strong positive correlation of expression with BMAL1.RT-PCR and Western blot showed that BMAL1 could promote IL-1R1.The ChIP assay and dual luciferase assay showed that BMAL1 combined with the IL-1R1 promoter and directly transcriptionally activated IL-1R1.(6)RT-PCR and Western blot showed that BMAL1 mediated the upregulation of cyclin D1 and IL-1R1 induced by IL-1β.Western blot also showed that overexpression of BMAL1 further aggravated the upregulation of cyclin Dl,IL-1R1,p-P65 and P65 caused by IL-1β.Western blot showed that BMAL1,cyclin D1,IL-1R1,p-P65 and P65 declined by treatment with IL-1R antagonist(IL-1RA),however BMAL1 overexpression partially relieved the decreasing effects of IL-1RA on cyclin D1,IL-1R1,p-P65 and P65.In rhythm-synchronized gastric epithelial cells,RT-PCR showed that IL-1β stimulation disrupted the rhythm of BMAL1,cyclin D1 and IL-1R1 expression,however the change of cyclin D1 and IL-1R1 disappeared after BMAL1 depletion.(7)In mouse model,the IHC expression of Ki67,BMAL1,P65,cyclin D1 and IL-1R1 upregulated with H.pylori and methyl-nitroso-urea(MNU)infection.Among another cohort of human specimens tested,the IHC expression of Ki67,BMAL 1,Ki67,P65,cyclin D1 and IL-1R1 was higher in cancerous tissues than that in adjacent normal tissues.In the correlation analysis of the relative values of IHC optical density,BMAL1 expression was positively correlated with Ki67,P65,CCND1 and IL-1R1.Conclusions1.We found that H.pylori up-regulated the expression of LIN28A,and LIN28A directly bound to the BMAL1 promoter,activating the transcription of BMAL1 to increase its expression.BMAL1 in turn promoted transcription of TNF-α by directly binding to the E-box elements on its promoter to increase its secretion.Therefore,our study revealed the mechanism through which disorder of circadian rhythm aggravated the inflammatory response induced by H.pylori,enriching the pathogenesis of H.pylori infection from a rhythmic molecular perspective.2.We found that H.pylori induced the expression of IL-1β,which bound to IL-1R1 on the surface of gastric epithelial cells,activated the intracellular NF-κB signaling pathway,and finally upregulated the circadian gene BMAL1 at the transcriptional level.BMAL1 in turn promoted IL-1R1 expression by binding to E-box elements of its promoter and made gastric epithelial cells more sensitive to IL-1βstimulus.Therefore a positive feedback loop between IL-1β/IL-1R1 and BMAL1 was formed,which augmented BMAL1 expression.The overexpressed BMAL1 facilitated abnormal proliferation of gastric epithelial cells by activating abnormal expression of cyclin D1.Therefore,our study revealed the role of BMAL1 in the conversion from gastritis to GC,enriching the mechanism of malignant transformation of gastric mucosa from the perspective of the clock gene BMAL1.