The Role And Mechanism Of Pattern Recognition Receptor NLRP3 And Histone Demethylase PHF8 In Gastric Carcinogenesis And Progression

Background:Gastric cancer(GC)is one of the leading causes of cancer death in less-developed countries.It is difficult to detect in the early stages and GC malignant progression is the main reason responsible for its poor prognosis.Based on data from Surveillance,Epidemiology,and End Results Program(SEER)18 2006-2012,the five-year survival rate of GC patients diagnosed with Stage I was up to 66.9%.However,more than 63%of GC patients were diagnosed with metastasis.Consequently,the five-year survival rate decreased sharply to 5%(stage IV).Therefore,it is critical to elucidate the mechanism of GC initiation and metastasis and to identify novel therapeutic approaches for GC.Helicobacter pylori(H.pylori)infection is the major cause of GC.H.pylori has been identified as a primary carcinogen by the World Health Organization/International Agency for Research on Cancer(WHO/IARC),and epidemiological data suggest that eradication of H.pylori infection can reduce the risk of gastric cancer.However,the mechanism of H.pylori induced carcinogenesis is still under investigation.Specially,there is a law worthy of attention:H.pylori infection has widely been suggested to trigger GC via an "inflammation-carcinoma chain"("chronic superficial gastritis(controllable inflammation)→ chronic atrophic gastritis with intestinal metaplasia(uncontrollable inflammation)→ atypical hyperplasia(precancerous lesions)→ gastric cancer ").Gastric mucosal malignant transformation is the result of the interaction of gastric environmental factors(external causes)with host regulatory responses(internal factors).H.pylori can recruit immune cells to gastric mucosal through MMP-2,9,CCL2 and initiate immune response,promoting the progress of inflammation to a chronic inflammatory,which is accompanied by the production of several proinflammatory cytokines,including IL-1,IL-6 and TNF-α,which play important roles in the tumorigenesis of chronic inflammation-associated cancer.Therefore,the "gastric area infection inflammation microenvironment" constituted by"H.pylori-gastric mucosal epithelial cells-immune cells" is the driver of gastric cancer development and progression.Pattern-recognition receptors(PRRs)are critical for recognizing invading microbial pathogens,generating mature pro-inflammatory cytokines(TNF-α,IL-6,IL-1β,etc),and triggering inflammation.Multiple PRRs,including Toll-like receptors(TLRs),nucleotide-binding oligomerization domain(NOD)-like receptors(NLRs),and retinoic acid-inducible gene(RIG)-I-like receptors(RLRs),are involved in gastric carcinogenesis.However,the role of NLRs in H.pylori infection and gastric tumorigenesis need more investigations.We searched the OncoMine database and found that NLRP3 expression was significantly increased in GC.NLRP3(a member of NLRs),also known as cryopyrin,PYPAF1 or Nalp3,has been implicated in many kinds of inflammation diseases and autoimmune diseases.NLRP3 inflammasome is closely associated with intestinal tumors,hepatocellular carcinoma and melanoma.NLRP3 polymorphism is also associated with GC risk.H.pylori infection can activate NLRP3 inflammasome and subsequent IL-1β secretion,indicating the potential roles of NLRP3 in H.pylori infection related diseases.The above findings suggest that H.pylori infection activated NLRP3 inflamasome enhances the risk of gastric cancer.However,the biological role of NLRP3 in GC progression remains unknown.It is clear that histone modification patterns alteration is involved in the development and metastasis of cancer.Other investigators also consider targeting histone demethylases as a new weapon in the fight against cancer.We performed a bioinformatics analysis of histone demethylase expression profiles from 876 GC patients using Kaplan-Meier Plotter.We found that high expression of two enzymes,KDM5C and PHF8,correlated with significant poor overall survival in GC.It has been reported that KDM5C promotes GC cells proliferation and invasion.Therefore,we focused on PHF8 and investigated possible link between enhanced PHF8 expression and GC progression.Serving as a transcription coactivator,PHF8(a JmjC-domain-containing protein)is a member of the histone demethylase family.Our study aim to clarify the regulatory mechanism of H.pylori induced histone demethylase PHF8 in gastric cancer progression.Aims1.Clarify the regulatory mechanism of Helicobacter pylori infection induced inflammation malignant transformation by promoting pattern recognition receptor NLRP3 expression.2.Clarify the regulatory mechanism histone demethylase PHF8 in gastric cancer progression.Methods and Results1.H.pylori infection markedly enhances NLRP3 expression through suppressing miR-22.Upregulated NLRP3 promoted inflammation malignant transformation through inflammsome-dependent and-independent functions,which triggered the cross-talk between gastric epithelial cells and macrophage.(1)We searched the OncoMine database and found that NLRP3 expression was significantly increased in GC.Western blot analysis confirmed that NLRP3 protein expression was markedly enhanced in human GC samples paired with adjacent non-tumor tissues,as well as in GC cell lines compared with GES-1,a normal gastric mucous membrane epithelial cell line.RT-PCR analysis confirmed that NLRP3 expression was significantly increased in human GC samples compared with AG tissues.Immunohistochemistry(IHC)staining showed that NLRP3 expression in superficial gastritis(SG)was low,and its expression was slightly upregulated in atrophic gastritis(AG)and dysplasia(DYS).But,NLRP3 expression was markedly enhanced in GC samples.In additional,survival times were lower in patients with high NLRP3 levels in different types of GC.Colony formation and xenograft model showed that enhanced NLRP3 expression promotes GC tumorigenisis.(2)Enhanced NLRP3 expression facilitates NLRP3 inflammasome assembly and subsequent IL-1β secretion.Colony formation assays and 5-ethynyl-2’-deoxyuridine(EdU)staining showed that IL-1β significantly increased colony formation of gastric epithelial cells.(3)Confocal showed that NLRP3 located in both cytoplasm and nucleus.Western blot showed that IL-1β greatly induced NLRP3 nucleic translocation in GC cells.Ensemble database suggested two potential NLRP3-binding sequences within CCND1 promoter region.qRT-PCR and Western-blot showed that NLRP3 overexpression also enhanced CCND1 protein expression and NLRP3 knockdown had the opposite effects.Dual luciferase assay and Chip assay showed that NLRP3 can bind with the promoter regions of CCND1.(4)By combining the microarray data(analyzed with AG and GC samples)with a bioinformatics search of Target-scan databases,we identified miR-22 was downregulated in GC and suggested miR-22 as a potential NLRP3-targeting miRNA.RT-PCR further confirmed that miR-22 expression was significantly downregulated in GC samples compared with AG samples,as well as in GC cell lines compared with GES-1,a normal gastric mucous membrane epithelial cell line.qRT-PCR and Western-blot showed that NLRP3 expression was dramatically decreased in miR-22-transfected BCG-23 and AGC cells,while miR-22 inhibitor had the opposite effects.Dual luciferase assay showed that miR-22 overexpression significantly decreased the luciferase activity with NLRP3 3’UTR,while miR-22 inhibitor greatly increased NLRP3-3’ UTR luciferase activity.(5)In macrophage,Western-blot showed miR-22 also markedly inhibited NLRP3 expression in THP-1 cells.Western-blot and ELISA showed that MiR-22 inhibits NLRP3 inflammasome activation and IL-1β secretion.(6)Xenograft model showed that miR-22 inhibits GC cells proliferation,as well as NLRP3 and CCND1 expression.(7)qRT-PCR and IHC assay found that higher level of NLRP3 expression was observed in the H.pylori-positive gastric tissues.qRT-PCR and Western-blot showed that H.pylori infection greatly enhanced NLRP3 expression in GC cell lines.Moreover,NLRP3 expression and NLRP3 inflammasome activation were also enhanced following H.pylori infection in THP-1 cells.H.pylori infection in N-methyl-N-nitrosourea(MNU)treated mice showed that H.pylori infection induced NLRP3 expression in vivo.In additional,H.pylori infection and IL-1β greatly inhibited miR-22 in both GC cells and THP-1 cells,then formed a positive feedback loop with NLRP3 expression,contributing to gastric carcinogensis.2.PHF8 interacts with β-catenin,and binds to the promoter region of vimentin,leading to the promotion of vimentin transcription and contributed to GC malignant progression.(1)By performing a bioinformatics analysis of histone demethylase expression profiles from 876 GC patients using Kaplan-Meier Plotter,we found that high expression of PHF8 correlated with significant poor overall survival in GC.Analysis of public microarray data from GEO repository,IHC and western-blot found that PHF8 expression was significantly increased in GC compared with adjacent normal tissues.Western-blot showed that PHF8 protein expression level was markedly increased in GC cell lines.(2)Animal experiment with subcutaneously injected GC cells into nude mice and injected GC cells into tail vein of nude mice showed that PHF8 promotes cell growth and metastasis of GC in vivo.(3)Clone formation,wound healing and transwell assays showed that PHF8 knockdown decreased proliferation and migration of GC cells,while PHF8 overexpression had the opposite effects,however,the catalytically inactive mutant PHF8 H247A overexpression reverse this effect.(4)Western-blot showed that PHF8 knockdown decreased the mesenchymal markers(Slug,ZEB-1 and vimentin)and increased the epithelial markers(E-cadherin and ZO-1).PHF8 overexpression had the opposite effects.In addition,PHF8 H247A mutant overexpression lost the regulatory effects on the expression of epithelial markers and mesenchymal markers in GC cells.Analysis of public microarray data from GEO repository showed that PHF8 expression was positively correlated with vimentin,ZEB-1 and Slug in 45 pairs of GC and corresponding normal tissues.qRT-PCR showed that PHF8 knockdown inhibited vimentin mRNA expression,while PHF8 overexpression enhanced vimentin mRNA expression.However,PHF8 H247A mutant overexpression abolished the enhancement of vimentin expression.Dual luciferase assay and Chip assay showed that PHF8 can bind with the promoter regions of vimentin.IP/Co-IP showed the interaction between PHF8 and β-catenin.(5)IHC staining showed that co-expression of vimentin and PHF8 existed in some GC samples and PHF8 expression was high in progressed GC and metastatic lymph nodes.Correlation analysis showed that PHF8 expression was positively correlated with vimentin expression,tumor size,invasion,lymph node metastasis.distant metastasis and TNM stage.(6)qRT-PCR and IHC assay found that higher level of PHF8 expression was observed in the H.pylori-positive gastric tissues.qRT-PCR showed that H.pylori infection greatly enhanced PHF8 expression in GC cell lines.H.pylori infection in N-methyl-N-nitrosourea(MNU)treated mice showed that H.pylori infection induced PHF8 expression in vivo.Conclusions1.We identified miR-22 as a suppressor of NLRP3 expression and NLRP3 inflammasome activation.MiR-22 has critical roles in maintaining the homeostasis of gastric microenvironments by inhibiting NLRP3 expression in both macrophages and gastric epithelial cells.H.pylori promotes gastric carcinogenesis by induced NLRP3 expression though inhibiting constituted expressed miR-22.Modulating NLRP3 expression by miR-22 may has significant implications for the intervention of GC.2.Our study proves that PHF8 is upregulated in GC and its overexpression is associated with proliferation and metastatic phenotype,predicting poor prognosis in GC patients.PHF8 binds to promoters of EMT related genes,such as vimentin,through interacting with P-catenin.Thus,we identify a novel mechanism for GC malignant preogression and suggest PHF8 as an attractive target for prevention and treatment of GC.