The Regulation Mechanism Of The Histone Chaperone CHAF1A And Histone Demethylase JARID1B In Gastric Carcinogenesis

Background:Gastric cancer(GC)is one of the most common malignant tumors,and its incidence rate ranks fourth in the world.Early diagnosis and intervention are beneficial to improve the cure rate of patients and prolong the survival of patients.Therefore,it is necessary to elucidate the molecular mechanisms of GC and explore the potential diagnostic,prognostic and therapeutic biomarkers for GC patients in China.Helicobacter pylori(H.pylori)infection is an oncogenic factor for gastric cancer.The infection rate of H,pylori in adults with high incidence of gastric cancer is above 60%in China.H.pylori interacts with host factors of gastric mucosa as a pathogenic environmental factor,induces the regulation of intracellular gene expression,and then increases the risk of gastric cancer.The complexity of carcinogenesis is not only caused by genetic alterations,but also involves epigenetic modifications.The role of epigenetic regulation in the development and progression of tumor has attracted more and more attention.The growing evidence has suggested that histone chaperones emerged as potential drivers in cancer initiation and progression.Histone chaperones are mainly involved in the process of folding,transporting,assembling and removing histones.In addition,they participate in the transcription or post-transcriptional regulation of gene by interacting with the transcription factors or epigenetic modification-related proteins,thereby regulating gene expression.And then affecting the cellular biological processes.The current study found that CHAF1A,as a core subunit of chromatin assembly factor-1(CAF-1),can regulate gene expression by interacting with histone methyltransferase SETDB1,histone deacetylase or the transcription factor Gfil.Recently,CHAF1A has been associated with the development and progression of solid tumors.By screening the expression of CHAF1A in the GEO database,we found that the expression of CHAF1A was up-regulated in gastric cancer tissues.This study aimed to explore the regulation mechanism of CHAF1A expression in gastric cancer and its biological function in the development of gastric cancer.Histone demethylase plays an important role in the tumor development and progression.JARID1B is a member of the JMJC histone demethylase family,which mediates the expression of cell cycle switch genes and regulates cell proliferation and differentiation by regulating the methylation of H3K4.JARID1B is up-regulated in many tumors,mediating tumorigenesis,invasion and metastasis.The current study found that JARID1B is up-regulated in gastric cancer,and our data analysis also found that gastric cancer patients with high expression of JARID1B had a poor prognosis.However,the mechanism by which JARID1B is up-regulated in gastric cancer is still unclear.This study focused to investigate the mechanism by which H.pylori promotes the expression of JARID1B and its function in the malignant transformation.Aims1.Clarify the role and regulation mechanism of histone chaperone CHAF1A in the gastric carcinogenesis.2.Clarify the regulation mechanism of histone demethylase JARID1B expression in gastric carcinogenesis.Methods and Results1.CHAF1A interacts with TCF4 to promote gastric carcinogenesis via upregulation of c-MYC and CCND1 expression.The transcription factor Spl regulates the expression of CHAF1A in gastric cancer cells at the transcriptional level,and H.pylori infection enhances CHAF1A expression through the transcription factor Spl.(1)We firstly carried out an analysis of GEO databases(e.g.GSE27342,GSE63089,GSE13195 and GSE2685)and found that the mRNA expression of CHAF1A was upregulated in GC tissues compared to adjacent normal tissues.Real-time quantitative PCR analysis showed that the expression of CHAF1A was also differentially expressed in several gastric cell lines,including GES-1,AGS,BGC-823,HGC-27,MGC-803 and SGC-7901.Of note,CHAF1A expression was higher in poorly differentiated HGC-27 and MGC-803 cell lines compared to immortalize epithelial GES-1 cells.Real-time quantitative PCR,Western-blot assay and immunohistochemical analysis showed that the expression of CHAF1A in gastric cancer was significantly higher than that in adjacent tissues.Kaplan Meier database analysis showed that CHAF1A overexpression was significantly associated with poor overall survival in patients of gastric cancer patients.(2)In vitro colony formation and CCK8 experiments found that CHAF1A knockdown reduced the proliferation of gastric cancer cells,while CHAF1A overexpression exhibited the opposite effects.Subcutaneous tumor formation in nude mice found that high expression of CHAF1A can promote the weight and volume of tumors in vivo.(3)The GEO database(GEO63089)showed that the expression of CHAF1A in gastric cancer tissues was positively correlated with the proliferation-related genes c-MYC and CCND1.The results of real-time quantitative PCR were consistent with the detection and analysis of clinical specimens.The results of real-time quantitative PCR and Western-blot showed that CHAF1A overexpression significantly increased the mRNA and protein expression levels of c-MYC and CCND1,while the depletion of CHAF1A demonstrated the opposite effects.(4)We found the interaction between CHAF1A and TCF4 through immunoprecipitation(IP)and co-immunoprecipitation in GC cells.The dual luciferase assay showed that TCF4 overexpression increased the luciferase activity mediated by Wnt signaling(TOP/FOP),whereas co-transfection of TCF4 and CHAF1A further enhanced their transcriptional activity.Consistently,silencing of CHAF1A significantly decreased luciferase activity.Finally,chromatin immunoprecipitation(ChIP)assays indicated the direct occupancy of CHAF1A on the promoter regions of c-MYC and CCND1.(5)The transcription factors binding to specific conservative elements were identified by PROMO 3.0.The promoter of CHAF1A containing multiple classical GGGCGG elements was recognized by transcriptional factor Sp1.Also,CHAF1A expression was positively correlated with Spl expression,indicated by GEO dataset(GSE63089)analysis of gastric tissues.The analysis of clinical specimens by real-time quantitative PCR verified the correlation between the expression of CHAF1A and Sp1.Real-time quantitative PCR and Western-blot experiments showed that the expression of CHAF1A was significantly down-regulated after Spl interference.The dual luciferase assay showed that knockdown of Spl significantly decreased the promoter activity of CHAF1A in GC cells,while Spl overexpression exhibited the opposite effects.Furthermore,ChIP analysis revealed that CHAF1A promoter region was occupied by Spl.(6)The analysis of microarray expression data from GEO dataset(GSE27411)showed that CHAF1A expression was significantly increased in H.pylori-infected gastritis tissues compared to non-infected tissues.The immunohistochemistry and real-time quantitative PCR analysis showed that CHAF1A was significantly higher in the gastritis specimens infected with H.pylori than in the uninfected.Real-time quantitative PCR and Western-blot experiments showed that H.pylori infection induced the expression of CHAF1A in gastric cancer cell lines.Real-time quantitative PCR detection of H.pylori-infected gastric cancer cell lines showed that H.pylori infection can promote the expression of CHAF1A,while Spl depletion abrogated the induction of CHAF1A in H.pylori-infected GC cells.2.H.pylori infection enhances JARID1B expression through the suppression of miR-29c,then promotes gastric cancer cell proliferation.(1)The Kaplan Meier database analysis showed that JARID1B overexpression was significantly associated with poor overall survival in patients of gastric cancer patients.(2)Real-time quantitative PCR revealed that the expression of JARID1B was up-regulated in gastritis tissues infected with H.pylori.H.pylori upregulates JARID IB expression of AGS and BGC-823 at different time points.Western-blot experiments also showed that H.pylori infection can promote the expression of JARID1B in cells.In the animal experiment of H.pylori combined with MNU treatment,the expression of JARID1B was slightly up-regulated in the low-dose MNU-treated group,and the expression of JARID1B was significantly up-regulated in the low-dose MNU combined with H.pylori-administered group.(3)The analysis of the TargetScan database showed that miR-29a/b/c can binds to the 3’UTR region of JARID1B.The expression of miR-29c in gastric cancer cells infected with H.pylori was down-regulated in GEO database(GSE51306),but the expression of miR-29b and miR-29c were not statistically significant.Real-time quantitative PCR results showed that the expression of miR-29c was down-regulated in gastritis tissues with positive H.pylori infection,and H.pylori infection could inhibit the expression of miR-29c in gastric cancer cells.(4)Western-blot results showed that miR-29c analogs(mimics)inhibited the expression of JARID1B,whereas the expression of JARID1B was up-regulated in gastric cancer cell lines transfected with miR-29c inhibitors.Real-time quantitative PCR detection revealed that miR-29c analogs can significantly inhibit the expression of JARID1B.The dual luciferase assay revealed that miR-29c analogs significantly inhibited the 3’UTR activity of JARID1B.Real-time quantitative PCR detection revealed that H.pylori infection could induce the up-regulation of JARID1B expression,while the transfection of the miR-29c inhibited the up-regulation of JARID1B expression.(5)In vitro EdU experiments and colony formation experiments showed that H.pylori infection can promote cell proliferation,interference with JARID1B can inhibit the proliferation of gastric cancer cells,and H.pylori infection combined with JARID1B interference can reduce the proliferation of gastric cancer cells.(6)Real-time quantitative PCR showed that the expression of CCND1 was significantly down-regulated after the interference with JARID1B in gastric cancer cell lines AGS and BGC-823.Analysis of the GEO database(GSE63089)showed that the expression of JARID1B was significantly positively correlated with CCND1.Conclusions(1)The histone chaperone CHAF1A is up-regulated in gastric cancer and promotes the proliferation of gastric cancer cells.CHAF1A acts on the c-MYC and CCND1 promoters by binding to the transcription factor TCF4 to promote malignant transformation of gastric mucosa.The expression of CHAF1A is regulated by the transcription factor Spl.Helicobacter pylori infection promotes the up-regulation of CHAF1A in gastric cancer through Sp1.Our study suggests that CHAF1A is a potential oncogene in GC,and may serve as a novel therapeutic target for GC treatment.(2)The overexpression of JARID1B is associated with poor prognosis in patients with gastric cancer.Helicobacter pylori can promote the expression of JARID1B in gastric cancer cells by inhibiting miR-29c.JARID1B can up-regulate the expression of CCND1 and promote the abnormal proliferation of gastric cancer cells.