Molecular Mechanism Of CREB-binding Protein Acetylation Induced By HBV Infection Regulating The High Expression Of TGF-β2

Research background and purposeHepatitis B virus(HBV)infection has a high incidence in China.Liver injury caused by HBV to cirrhosis and even liver cancer needs to undergo liver fibrosis.The occurrence of liver fibrosis and fibrosis-related liver cancer worldwide is also the reason for the increase in morbidity and mortality.Early treatment can reverse liver fibrosis,which is crucial for the correct understanding of the mechanism of liver fibrosis caused by HBV.Acetylation of proteins has a variety of physiological functions,such as transcriptional regulation,metabolic regulation,signal transduction regulation,etc.,and also regulates a variety of properties of proteins,such as protein stability,subcellular localization,transcriptional activity,etc.Compared with the control group without HBV infection,HBV infection can induce the change of protein acetylation level in liver cancer cells,indicating that this effect is caused by HBV infection.In this study,mass spectrometry was used to detect the acetylation modification of HBV-infected and non-infected liver cancer cell lines.The acetylation modification protein CREBBP(CREB-binding protein)enriched in TGF-β pathway related to liver fibrosis was screened by differential acetylation modification protein analysis.The molecular level verification showed that HBV infection could effectively up-regulate TGF-β2 level and inhibit HBV infection to down-regulate TGF-β2 level.In addition,knocking down CREBBP under HBV infection can downregulate TGF-β2 level.This study demonstrated that HBV infection up-regulated CREBBP acetylation and promoted the high expression of TGF-β2,thereby promoting the development of liver fibrosis.We have revealed the molecular mechanism of liver fibrosis caused by HBV infection and provided new ideas for clinical treatment of liver fibrosis.Methods1.The acetylated proteins were extracted from HepG2-NTCP cells and HBV infected HepG2-NTCP cells.The acetylated proteins were screened by modification enrichment technology and database search.2.Protein annotation,motif analysis of modification sites and protein function enrichment analysis of these proteins were performed to roughly determine the location and function of these acetylated modification proteins in cells.3.Acylated proteins enriched in TGF-β pathway were obtained by KEGG pathway enrichment analysis.4.The effect of the selected acetylated modification protein on the TGF-β pathway was verified by experiments,that is,the target protein gene was knocked out by siRNA,and the expression level of TGF-β2 was detected by qRT-PCR.ResultsThe mass spectrometry analysis showed that the protein differential modification analysis showed that the protein acetylation modification level changed in HBV-infected liver cancer cells,with 22 significantly up-regulated and 77 significantly down-regulated protein acetylation modification sites.In the number of protein acetylation modifications,20 were up-regulated and 67 were down-regulated.Functional classification of differential modification proteins,including GO secondary annotation classification and subcellular structure localization classification,showed that most of the differential modification proteins were located in cytoplasm and nucleus,involved in cell process,and had binding and catalytic activity.GO enrichment analysis of differentially modified proteins showed that the enriched GO items included peptide-lysine-N-acetyltransferase activity,histone acetyltransferase activity,peptide-N-acetyltransferase activity,activated transcription factor activity,N-acetyltransferase activity,N-acyltransferase activity,hormone receptor binding,DNA binding in transcriptional regulatory region and nucleic acid binding regulatory region.The enrichment analysis of differential modified proteins by KEGG pathway showed that the most abundant KEGG items included Notch signaling pathway,JAK-STAT signaling pathway,renal cell carcinoma,thyroid hormone signaling pathway,and TGF-β signaling pathway,among which JAK-STAT signaling pathway and TGF-β signaling pathway were related to liver fibrosis.The protein domain aggregation analysis of the differentially modified proteins showed that the significantly enriched protein domains included zinc finger structure,phosphopanthionylethylamine-binding ACP domain,nuclear receptor coactivator,CBP/p300 atypical cyclic domain,CBP/p300 histone acetyltransferase domain,KIX domain CBP coactivator,histidine kinase-like ATPase(C-terminal domain),bromine domain,and histone folding domain.The enrichment analysis of TGF-β signaling pathway showed that CREBBP was enriched in TGF-β signaling pathway and acetylated at amino acids 434 and 439.By detecting the expression of TGF-β2 in liver cancer cells infected with HBV and infected with siRNA targeting CREBBP,qRT-PCR was used to detect the expression level of TGF-β2 in cells.It can be concluded that HBV regulates the high expression of TGF-β2 by inducing CREBBP acetylation.Conclusion1.HBV-infected hepatocellular carcinoma cells induce CREBBP acetylation expression;2.CREBBP acetylation regulates the high expression of TGF-β2.