Stat1is Transcriptionally Down-regulated By HSF4 In Hepatoma Cells SMMC-7721

Objective:STAT1 plays an essential role in innate immunity by protecting the host from virus infections and other pathogens.STAT1 has both pro-apoptotic and anti-proliferative activities in tumor cells,plays important roles in cell signaling processes.However,the expression regulation of STAT1 in hepatocellular carcinoma are still poorly understood.To characterize the mechanisms that control STAT1 expression and its down-regulation in cancer,we used bioinformatics software to analyze the transcription factors that might bind to the STAT1 promoter region,the correlation between HSF4 and STAT1 expression was found.RNAi silencing of HSF4 in SMMC-7721 elevated STAT1 mRNA and protein expression.Analysis followed by ChIP identified HSF4 binding sites in promoter region from 254-489 bp upstream the transcription start site(TSS)of STAT1 gene,the predicted binding site is just within this region and was verified by the following EMSA experiments.Promoter activity of stat1 was determined by luciferase reporter assay when co-transfected with HSF4.The decrease of cell proliferation and increase of cell apoptosis were observed after RNAi silencing of HSF4 in SMMC-7721.Our results identify stat1 as a novel transcriptional target of HSF4 in SMMC-7721 cell line,thus provide a basis for the development of therapeuticstrategies for hepatocellular carcinoma.At present,the research on stat1 mainly focuses on its function.There is no report about the regulation mechanism of stat1 in the literature.It is helpful to reveal the mechanism of stat1 in revealing tumorigenesis.In this study,the regulation mechanism of stat1 from the transcription factor level was studied.Research methods:(1)Cell culture The human liver normal cell line L-02 and the human hepatocellular carcinoma cell line SMMC-7721 were cultured in RPMI1640 medium(containing 10% calf serum)and cultured in a 37 °C.incubator with 5% CO 2(2)Transcription factor prediction screening The bioinformatics software JASPAR was used to predict the transcription factors that may bind to the stat1 promoter region.The predicted transcription factors were screened to find candidate genes.(3)HSF4 siRNA interference and high expression establishment for cell line Lentivirus method: the HSF4 siRNA interference fragment of HSF4 interferes with the SMMC-7721 cell line The complete gene sequence of HSF4 was cloned into carrier p EX-4 to form PEX-4-HSF4 expression plasmid.(4)Real-time PCR detection of mRNA expression Real-time PCR was used to detect the expression of HSF4 and stat1 in SMMC-7721 cells and the mRNA expression of HSF4 and stat1 before and after siRNA interference.(5)Western blot Western blot was used to detect the protein expression of HSF4 and stat1 before and after siRNA interference with SMMC-7721 cell line.(6)Ch IP-sequencing The complete genomic DNA was extracted to make ChIP-sequencing,and require the whole genomic DNA combined with HSF4 in SMMC-7721 cell line.The CHIP-seq is provided to the HSF4 binding area,which is compared with the sequence of JESPAR predicated result to find out the binding site between stat1 and HSF4.(7)Luciferase reporter gene The luciferase reporter gene plasmid and hsf4 gene expression plasmid were transfected into hek293 cells,and the expression of luciferin was observed before and after transfection.(8)Apoptosis experiment Pe-7ADD apoptosis reagent/flow cytometry was used to detect the apoptosis of SMMC-7721 cells before and after HSF4 interference.(9)The experimental data were statistically processed The data were analyzed by image acquisition and analysis system.The data was expressed as mean ± standard deviation(± s).Using statistical software,single-factor analysis of variance was used to compare groups.P <0.5 was considered statistically significant,P <0.01 was Significant statistical significance.Results: First,Bioinformatics software predicts transcription factor binding sites the transcription factor HSF4,a transcription factor that can bind to the stat1 promoter region,was predicted by bioinformatics software JASPAR.Second,detect the correlation between HSF4 and stat1 expression we carried out realtime PCR and western blot on HSF4 and stat1,and found that the expression of HSF4 was opposite to the expression of stat1 in SMMC-7721 cell line.Third,siRNA interference method to detect HSF4 regulation of STAT1 expression siRNA interference HSF4 SMMC-7721 cell line,Realtime PCR,Western Blot detection and found that stat1 in SMMC-7721 cell line increased,suggesting that HSF4 STAT1 expression regulation Fourth,ChIP-sequencing Bioinformatics software was used to predict and determine the transcription factor HSF4,then make further HSF4 chip-s,and in the promoter region found stat1 binding site.Fifth,luciferase reporter gene the specific sequence binding of transcription factor HSF4 and its promoter STAT1 was examined.The change activity of promoter STAT1 was detected by knocking out the transcription factor HSF4.Sixth,apoptosis and value-added experiments progress through biological experiments to verify the regulation of the transcription factor HSF4 and the promoter STAT1 relationship.Conclusion: It is preliminary evidence that the transcription factor HSF4 in human hepatocellular carcinoma cell line SMMC-7721 regulates the expression of STAT1 and find the binding region of HSF4 and STAT1.