Mechanism Research On Human IRF-3 Gene Transcriptional Regulation

Backgrounds:Interferon regulatory factor 3(IRF-3)is a member of the family of interferon regulatory factors and is primarily responsible for the induction of type I interferon(IFN),interferon-stimulated genes(ISG)and pro-inflammatory cytokines,playing an important role in innate immunity.However,recent studies have found that IRF-3 not only participates in the innate immunity,but its abnormal expression in cancer also brings new ideas for cancer research.However,little is known about the transcriptional regulation of IRF-3 in lung cancer.Early studies found that the transcription factor GATA-1 can promote the differentiation and maturation of erythrocytes and play an important role in the hematopoietic function in the embryo.Later studies found GATA-1 activates or inhibits the transcription of genes in erythrocytes,megakaryocytes,mast cells,and neutrophils.The GATA-1 protein has two highly conserved zinc finger domains.The zinc finger domain at the C-terminus can mediate GATA-1 binding to the WGATAR sequence of the downstream gene.The acetylation level of this domain can be regulated by histone acetyltransferase inhibitor(HDACi)and some transcriptional activating factor,like p300,altering the transcriptional properties of GATA-1 to gene promoters.Objective:The aim of this study is to explore the abnormal expression of IRF-3 and GATA-1 in human lung adenocarcinoma and paracancerous tissues,analyze the core promoter region of IRF-3 gene promoter,identify the key transcription factors that regulate the expression of IRF-3 in lung adenocarcinoma cells.This study also aims to explore the regulation of HDACi on IRF-3 promoter in lung adenocarcinoma cells and to explore the role of IRF-3 in HDACi associated adjuvant treatment for lung adenocarcinoma.Methods:The online bioinformatics software TFSEARCH and Matlnspector were used to identify the transcriptional factor binding site of the DNA fragment from-149 bp to +18 bp in IRF-3 promoter.Based on the prediction results of bioinformatics,Primer 5.0 design software was used to design the primers delete GATA-1 binding site on the IRF-3 promoter.The effect of GATA-1 on IRF-3 promoter was detected by luciferase activity of a deletion mutation of the GATA-1 binding site on IRF-3 core promoter luciferase plasmid.ChIP and EMSA were demonstrated to verify the in vivo and in vitro binding of the transcription factor GATA-1 on the IRF-3 promoter in lung adenocarcinoma A549 cells before and after the TSA stimulation.Using the results of Oncomine database,we analyzed the expression of IRF-3 and GATA-1 in lung adenocarcinoma versus paracancerous tissues,and the relationship between IRF-3 and GATA-1 expression.The expression of IRF-3 and GATA-1 in lung adenocarcinoma specimens was detected by qPCR and Western Blot.Small interfering RNA and transcription factor overexpression assays were detected to identify the transcriptional regulation of IRF-3 by GATA-1.The promoter luciferase activity,the mRNA and protein of IRF-3 was investigated before and after the treatment of TSA.GATA-1 binding site deletion promoter was also detected before and after TSA stimulation.These experiments were to demonstrate the effect of TSA on IRF-3 transcriptional activity.The expression of GATA-1 in A549 cells was localized by immunofluorescence.Use immunoprecipitation method to verify the acetylation of GATA-1 stimulated by TSA.ChIP-qPCR method was to detect the binding of GATA-1 on IRF-3 promoter.Overexpression of p300 was performed to observe its effect of on IRF-3 promoter activity and mRNA expression,and the relationship between p300 and GATA-1 binding on the promoter was analyzed.Results:By bioinformatics analysis we obtained that the core region of the human IRF-3 promoter lies between-67 bp and-149 bp(the transcription start site is set as+1);transcription factors GATA-1,HSF,CdxA,and E2F1 are possible to modulate the transcription of the human IRF-3 gene by interacting with this core promoter region.After deletion mutation of GATA-1,we found that the IRF-3 promoter luciferase activity is dependent on the GATA-1 binding site.Using ChIP and EMSA techniques,we confirmed that GATA-1 can bind to the IRF-3 core promoter in A549 cells via both in vivo and in vitro pathways.The statistics of Oncomine database showed that the mRNA expression of IRF-3 in patients with lung adenocarcinoma was significantly higher than that in paracancerous tissues,while the expression of GATA-1 mRNA was significantly lower than that in adjacent tissues,which was consistent with the expression of IRF-3 and GATA-1 protein levels in lung adenocarcinoma tissues and paracancerous tissues we obtained.The knockdown of intracellular GATA-1 by siRNA and overexpression of GATA-1 confirmed that GATA-1 negatively regulates the transcriptional regulation of IRF-3 in lung adenocarcinoma cell line A549.TSA and VPA upregulated the activity of IRF-3 promoter and mRNA expression.After deletion mutation of GATA-1 binding site on the IRF-3 promoter,TSA could not upregulate the activity of the IRF-3 promoter.Immunofluorescence detected GATA-1 expression in the nucleus.ChIP-qPCR and immunoprecipitation experiments demonstrated that TSA stimulated acetylation of GATA-1 and enhance its binding to the IRF-3 promoter.TSA also stimulates the binding of p300 to the IRF-3 promoter,enhancing the activity of the IRF-3 promoter.Conclusion:Oncomine database and experiments showed that the expression of IRF-3 and GATA-1 was negatively correlated in lung adenocarcinoma,and the up-regulation of IRF-3 may be related to the pathogenesis of lung adenocarcinoma.2.The core promoter region of human IRF-3 gene is located between-67 bp and-149 bp.GATA-1 can regulate the promoter activity of IRF-3 through its in vivo and in vitro binding to this region and down-regulate IRF-3 mRNA transcription activity.3.TSA and VPA upregulate the promoter activity of IRF-3,and this up-regulation effect depends on the binding of GATA-1 on the IRF-3 promoter.4.TSA induced increasing acetylated GATA-1 binding with IRF-3 promoter.