The Study Of Effects And Regulatory Mechanism Of Reg3g/FOXM1 On Dendritic Cell In Tumor Environment

BackgroundDendritic cells(DC),critical regulators of host immunity,serve as a bridge between innate and adaptive immune response.During initiation and tumorigenesis,the maturation and function of DC are suppressed by tumor environment construed by tumor,tumor-associated cells and their secretions,nor does the immune response.We found that Reg3g overexpression suppressed the maturation of dendritic cells(DCs),accelerating interactions of PD-1 and PD-L1,thereby restraining cytotoxic activities of CD8~+T cells and producing a tumor environment consisting of Th2 cytokines IL-10,TGF-?,consequently accelerating tumor growth.ObjectiveTo investigate the molecular mechanism and crucial gene of Reg3g on maturation and function of DCs in Pac tumor environment.Then identify the expression and effects of the crucial gene on DCs in tumor environment.After that,explore the epigenetic regulatory mechanism of H3K79me2 modification on the crucial gene in DCs.MethodsFirstly,we retrieved the datasets from the NCBI GEO databank in order to detect mRNA levels of REG3A and DC-associated genes in the pancreatic tumors tissues.Afterwards we obtained DCs from ectopic mouse model of Pac.The phenotypes and EGFR expression in Reg3g treated DCs were detected separately by FACS and immunofluorescence.Then,Erlotinib was used to identified the essential role of EGFR in Reg3g activated STAT3 pathways by Western Blot.After that,we identified the gene affected in DCs of Reg3g treatment by analyses of NCBI GEO databank,Gene Ontology functions,KEGG pathways,Literature retrieval and qRT-PCR.Lastly,we detected the effects of Reg3g treated DCs conditioned medium on tumor cell proliferation by MTT assays.Secondly,we determined the importance of the crucial gene in DCs under tumor environment in GEO Database and STRING Database.Afterwards we obtained DCs from ectopic mouse model of pancreatic and colon cancer.The maturation and function of DCs as well as the crucial gene expression were detected.Then Thiostrepton was used to identify the role of the crucial gene in DCs maturation and function.Thirdly,we detected the enrichment of histone modification at the promoter of the crucial gene in various tissues and cell lines in ENCODE Database,and we also investigated the correlation between histone methyltransferases and the crucial gene in GEO Database.Then we examined the effects of EPZ,an inhibitor to DOT1L,on pancreatic,colon cancer cells and DCs(ectopic and orthotopic model or tumor conditioned medium).By using ChIP,qRT-PCR,Western Blot and FACS,we not only detected the enrichment of H3K79me2 at the promoter of the crucial gene in tumor cells and DCs,but also tested the CCNA2,CCNB1 expression and proliferation of cancer cells as well as maturation and function of DCs.After that,we identified the target gene in DCs of the crucial gene by analyses of Gene Ontology function,KEGG pathways and Literature retrieval.Then,the combination of the crucial gene on the promoter of candidate and its mRNA expression were determined separately by ChIP and qRT-PCR.Lastly,we detected the effects of DCs conditioned medium on H3K79me2 modification at the promoter of the crucial gene in tumor cells.ResultsFirstly,with the datasets from NCBI GEO databank,we found REG3A and TGFB1 increased,while the CD86,IFNG,IL-12A decreased in pancreatic cancer tissues compared to the normal ones.The expression of ITGAX seemed no difference between tumor tissues and normal ones.The treatment of Reg3g increased the EGFR expression in DCs from ectopic pancreatic model,so did the pSTAT3.EGFR interference with Erlotinib decreased both expression of EGFR and pSTAT3 in DCs.With analyses of microarray data and bioinformatics,we next screened FOXM1 as a candidate.The Reg3g treatment upregulated FOXM1 in DC.At last,we found the conditioned medium form Reg3g treated DCs promoted tumor cell proliferation.Secondly,with GEO Database,we found FOXM1 increased in immature DCs and tumor serum cultured DCs.FOXM1 was observed to connect with 12 other genes in the protein-protein association network,suggesting an essential role of FOXM1 in DCs maturation in tumor environment.In DCs from ectopic mouse model,we found MHC-II,CD86 and CCR7 decreased,while PD-L1 increased.The IL-12p70production and promotion to T cell proliferation of DCs were attenuated,accompanied with an increased level of FOXM1.Furthermore,inhibiting FOXM1up-regulated CD86 and CCR7,but lowered PD-L1 on the DCs surface.The inhibition also increased IL-12p70 production and promoted T cell proliferation.Additionally,high expression of FOXM1 in DCs correlated with poor survival in pancreatic and colon cancer patients.Thirdly,with ENCODE and GEO Datasets,we observed the promoter of FOXM1 was enriched with H3K79me2 in various cell lines and tissues.Besides,we found that only expression of DOT1L,the sole enzyme that specificity mediates H3K79 methylation,correlated with FOXM1 in both pancreatic and colon cancer tissues.Inhibition of H3K79me2 modification not only attenuated the tumor cell viability,but also reduced the H3K79me2 enrichment at the FOXM1 promoter,resulted in decreasing FOXM1 and its downstream genes CCNA2,CCNB1expression.Increased H3K79me2 enrichment was observed at the FOXM1 promoter in both DCs from tumor bearing mice,and DCs from wild-type mice cultured with tumor conditioned medium which mimicked the tumor microenvironment(TME).Furthermore,inhibiting H3K79 methyltransferase DOT1L activity not only decreased the enrichment of H3K79me2,but also downregulated expression of FOXM1 and partially reversed its immune suppressive effects on DCs.Furthermore,we screened a potential target gene WNT5A with analyses of microarray data and bioinformatics.We found the increased combination of FOXM1 on the promoter of WNT5A in DCs from ectopic and orthotopic model of pancreatic and colon cancer,so did the mRNA expression of WNT5A.Additionally,inhibition of both H3K79me2 and FOXM1suppressed WNT5A expression.ConclusionFirstly,in Pac tumor environment,Reg3g suppresses the maturation and function of DCs,accompanied with an increased level of FOXM1,suggesting that a potential role of FOXM1 in regulation of DCs in tumor environment.Secondly,FOXM1 overexpression inhibits DCs maturation and function in pancreatic and colon cancer tumor environment.Thirdly,H3K79me2 modification up-regulates the transcription of FOXM1.Increased FOXM1 not only promotes CCNA2 and CCNB1 expression in tumor cells,but also activated WNT5A in DCs,suppresses the maturation and function of DCs in tumor environment.