Protein Acetylation Increased Risk of Fibrosis-Related Liver Cancer

Objective The occurrence of liver fibrosis and fibrosis-related liver cancer is the reason for the increase in morbidity and mortality worldwide. Transforming growth factor-β2 (TGF-β2) is an important mediator of chronic liver fibrosis. This study aims to find the molecular mechanism that mediates HBV infection and induces TGF-β2 and verifies that CREB binding protein acetylation mediates HBV infection and induces TGF-β2 expression. Methods The acetylated proteins were extracted from HepG2-NTCP cells and HBV-infectedHepG2-NTCP cells. The acetylated proteins were screened by modification enrichment technology and database search. Protein annotation, motif analysis of modification sites, and protein function enrichment analysis of these proteins were performed to roughly clarify the location and function of these acetylated modification proteins in cells. Acylated proteins enriched in the TGF-β pathway were obtained by KEGG pathway enrichment analysis. 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 the TGF-β2 was detected by qRT-PCR. Results Proteins were extracted from HepG2-NTCP cells and HepG2-NTCP cells infected with HBV, and differential acetylation modification proteins were screened. The target protein CREB binding protein was screened by modification enrichment technology and database search. The aggregation analysis of TGF-β pathway showed that CREB binding protein was acetylated at amino acid positions 434 and 439, and enriched in the TGF-β signaling pathway. siRNA targeting CREB binding protein was transfected, and the expression of TGF-β2 in cells was detected by qRT-PCR and western blot, respectively. It was verified that HBV infection-inducedCREB-binding protein acetylation regulated the high expression of TGF-β2. Conclusion After HBV infection, CREBBP acetylation was up-regulated, which promoted the high expression of TGF-β2.


Introduction
Liver fbrosis can develop into chronic hepatitis and cancer of liver and was the main incentive of various liver diseases [1]. Progressive liver fbrosis and tumors have many causes, including non-alcoholic fatty liver disease, viral hepatitis, alcoholism, autoimmune hepatitis, nonalcoholic steatohepatitis (NASH), and biliary tract diseases [2]. Liver fbrosis was closely related to cancer. Te incidence of hepatocellular carcinoma caused by liver cirrhosis is as high as 90%, which makes cirrhosis a major risk factor for liver cancer. Te only efective treatment of liver fbrosis was eliminating irritation or liver transplantation. It was necessary to become potential antiliver fbrosis treatment methods to reduce fbrosis and the risk of liver cancer [3].
TGF-β2 always exists in all stages of the hepatic lesion [4]. Te initial stage of the lesion induces hepatocyte apoptosis and HSCs transdiferentiation into myofbroblasts after chronic injury. TGF-β2 also promotes HSC proliferation and maintains myofbroblast phenotype, which is the key to the formation of liver cirrhosis. TGF-β also plays an important role in hepatocellular carcinoma; it acts as a tumor suppressor at the early stage, but once tumor cells gain the ability to overcome their cytostatic response, it activates key tumor-promoting factors. Te role of cytokines is conducive to malignant progression [5].
Various causes of liver disease lead to liver fbrosis through a comprehensive signal network that regulates the deposition of extracellular matrix. Te hepatitis B virus (HBV) is still the leading cause of liver fbrosis in China [6]. More and more evidence indicates that HBV infection may promote the production of transforming growth factor-β in liver cells, which in turn activates hepatic stellate cells and accelerates liver fbrosis [7,8]. TGF-β is mainly produced by activated macrophages in the liver, which stimulates the activation of hepatic stellate cells (HSCs) into a myofbroblast-like phenotype [9,10], promotes the diferentiation of myofbroblasts, and stimulates the synthesis of extracellular matrix and down-regulating the degradation of extracellular matrix [11]. It is reported that TGF-β2 binds to the type II receptor on the cell surface, then recruits the type I receptor. TGF-βI receptor activates Smad2 and Smad3 proteins, and the activated Smad2 and Smad3 proteins specifcally bind to each other. Te binding protein complexes are phosphorylated and then bound to Smad4 proteins, and then transported to the nucleus to the binding to DNA, and regulate the transcription of extracellular matrix genes in the nucleus. In addition, studies have shown that Smad2 and Smad3 may also participate in the general transcription mechanism through direct interaction with p300 and CREBBP, and may participate in the development of liver fbrosis through the transcription of extracellular matrix [12].
Te results have shown that CREB binding protein is involved in many cellular processes and functions. CREB binding protein interacts with a variety of transcription factors, including CREB binding protein and a variety of nuclear hormone receptors, to play the role of transcription co-activator and histone acetyltransferase [13,14]. Studies have found that CREB binding protein is related to fbrosis, inhibiting Wnt/β-catenin/CREB binding protein signal transduction and reversing pulmonary fbrosis [15], Grap2 cyclin D interaction protein negatively regulates CREB binding protein and inhibits fbroblast-like synovial cell proliferation [13]. Currently, it was limited research on the role of CREB-binding protein in liver fbrosis. Inhibition of CREB binding protein/β-catenin can inhibit the formation of liver fbrosis and promote the regression of liver fbrosis [16]. In addition, CREB binding protein is involved in the signal pathway of hypoxia-induciblefactor-1α (HIF-1α), erythropoietin. Tese signaling pathways are activated during cerebral ischemia and exert neuroprotection [17]. Everyone has gradually realized the important relationship between TGF-β and fbrosis. Here, we have also proved that HBV infection up-regulates the acetylation level of CREB binding protein and induces high expression of TGF-β2 (Figure 1), which provides a new treatment for liver fbrosis.
Here, we report that the acetylation level of CREB binding protein is signifcantly up-regulated in HBVinfected hepatocytes and CREB binding protein mediates liver fbrosis through the TGF-β pathway.

Protein Preparation.
Te sample was sonicated three times on ice using a high-intensity ultrasonic processor (Scientz) in lysis bufer (8 M urea, 1% Protease Inhibitor Cocktail) (Note: For PTM experiments, inhibitors were also added to the lysis bufer, e.g., 3 μM TSA and 50 mM NAM for acetylation). Te remaining debris was removed by centrifugation at 12,000 g at 4°C for 10 min. Finally, the supernatant was collected and the protein concentration was determined with a BCA kit according to the manufacturer's instructions.

Protein Diferential Modifcation
Analysis. Te dithiothreitol was added to the protein solution to a fnal concentration of 5 mM and reduced at 56°C for 30 min. Ten add iodoacetamide to make the fnal concentration 11 mM, and incubate for 15 min at room temperature in the dark. Finally, the urea concentration of the sample is diluted to less than 2 M. Add pancreatin at a mass ratio of 1 : 50 (pancreatin: protein) and digest overnight at 37°C. Ten add pancreatin at a mass ratio of 1 : 100 (pancreatin: protein) and continue enzymatic hydrolysis for 4 h. Te signal abundance corresponding to the modifed site in each sample was detected by mass spectrometry [18]. According to the strength of the modifed peptide, strength of the modifed peptide in each sample was obtained by nonstandard quantitative calculation.  acetylated resin that has been washed in advance medium (antibody resin item number PTM104, from Hangzhou Jingjie Biotechnology Co., Ltd., PTM Bio), placed on a 4°C rotating shaker, gently shake and incubate overnight. After the incubation, the resin was washed 4 times with IP bufer solution and twice with deionized water. Finally, use 0.1% trifuoroacetic acid eluent to elute the resin-bound peptides, eluting three times, collecting the eluent, and vacuum freeze and drain. After draining, follow the instructions of C18 ZipTips for desalination, vacuum freeze draining, and then use for LC/MS analysis.

Motif Analysis.
Soft MoMo (motif-x algorithm) was used to analyze the model of sequences constituted with amino acids in specifc positions of modify-21-mers (10 amino acids upstream and downstream of the site, but phosphorylation with modify-13-mers that 6 amino acids upstream and downstream of the site) in all protein sequences. And all the database protein sequences were used as background database parameters. Te minimum number of occurrences was set to 20. Emulate original motif-x was ticked, and other parameters with default.

Histological Analysis and Immunohistochemistry.
Parafn sections of human liver tissues were prepared by hematoxylin-eosin (H & E) staining. Te parafn sections were subjected to immunohistochemical detection of TGF-β2 and observed under a microscope (Olympus BX51, Japan). Image J software was used to quantitatively analyze TGF-β2.

Sample Repeatability Test.
Repeatability experiments were used to verify the validity of the experiment. Compared with the control group, the experimental group was transfected with HBV-expressed HBsAg (Figure 2(a)). Te effectiveness of this experiment is proved by using three statistical analysis methods: principal component analysis( Figure 2

Analysis of Acetylated Modifed Proteins.
Trough protein motif analysis of the proteins expressed by hepatocytes after HBV infection, the acetylated modifed proteins were screened. Totally, 450158 secondary spectra were got by mass spectrometry. Te secondary spectra of MS were retrieved from the protein theory database. Te number of available efective spectra was 70922, and the utilization rate of spectra was 15.8%. A total of 16487 peptides and 5958 acetylated peptides were identifed by spectral analysis. We have identifed 6065.0 acetylation modifcation sites on 2595.0 proteins, of which 4168.0 sites on 1988.0 proteins have quantitative information (Figure 3(a)). Trough protein diferential modifcation analysis, 22 acetylation sites were up-regulated in HBV-transfected liver cancer cells, 77 acetylation sites were down-regulated, and 20 acetylationmodifed proteins were up-regulated. Te levels of two acetylated modifed proteins were down-regulated ( Figure 3(b)). Figure 2(c) shows the distribution of these diferential proteins in the volcano map of diferential modifcation sites (Figure 3(c)).
Protein motif analysis calculates the regular trend of the amino acid sequence in the region where the acetylation modifcation site occurs by counting the rules of the amino acid sequence before and after all acetylation modifcation sites in the sample. Tis analysis can fnd the sequence characteristics of modifcation sites, and thus speculate or determine the enzymes related to modifcation (Figure 4(a)). In addition, gene ontology also explains the biological efects of proteins from three perspectives: biological process, molecular function, and cell composition. Most of the proteins corresponding to acetylation modifcation sites are distributed in cells and participate in cellular processes. Exerting molecular functions such as binding, structural molecular activity, transcription factor activity, catalytic activity, and molecular function modifer (Figure 4(b)). Among them, 39 proteins were distributed in the cytoplasm, accounting for 44.83%, 34.48% in the nucleus, and the rest in mitochondria, plasma membrane, extracellular, and so on (Figure 4(c)).

Functional Enrichment Analysis of Proteins Corresponding to Diferent Acetylation Modifcation Sites.
Te bubble chart of the enrichment distribution in the GO classifcation shows that the diferential protein has acetyltransferase activity, hormone receptor activity, and DNA binding function in the transcription regulatory region ( Figure 5(a)) (protein function: peptide-lysine-Nacetyltransferase activity, histone acetyltransferase activity, peptide-N-acetyltransferase activity, activating transcription factor activity, N-acetyltransferase activity, Nacyltransferase activity, p53 binding, core promoter proximal DNA binding, nuclear hormone receptor binding, hormone receptor binding, sequence-specifcdoublestranded DNA binding, RNA polymerase II regulatory region DNA binding, transcription coactivator activity, double-stranded DNA binding, nucleic acid binding transcription factor activity, chromatin binding, sequence specifc DNA binding, transcription regulatory region DNA binding, nucleic acid binding regulatory region, regulatory region DNA binding). Te enrichment distribution of the proteins corresponding to the diferential acetylation modifcation sites in the KEGG pathway shows that two interesting proteins are enriched in the TGF-bate signaling pathway (P < 0.002) ( Figure 5(b)). Diferential acetylation modifcation sites correspond to protein enrichment distribution in protein domain classifcation. Te bubble chart shows that diferential proteins are enriched in CBP/p300 atypical loop domains and CBP/p300 histone acetyltransferase domains ( Figure 5(c)).
In conclusion, acetylated proteins were diferentially expressed in HBV-infected HCC cells, and the corresponding proteins at diferent modifcation sites were highly enriched in the TGF-β signaling pathway. In the domain classifcation of diferential modifcation proteins, the strong aggregation was concentrated in the CBP/p300 atypical cyclic domain; diferentially modifed proteins have transcriptional regulatory functions.

Enrichment of Diferentially Acetylated Protein CREBBP
in TGF-β Pathway. CREB binding protein is enriched in the TGF-β signaling pathway. Further enrichment analysis of the TGF-β signal transduction pathway, as shown in Table 1, the CREB binding protein was acetylated at amino acid positions 434 and 439 and was enriched in the TGF-β signal transduction pathway.

HBV Infection Up-Regulates CREBBP Expression and
Induces High Expression of TGF-β2. Te TGF-β2 expression level gradually increased within 3 days after the HepG2-NTCP cell line was transfected with HBV, and it was positively correlated with HBsAg (Figure 7(a)). After preventing HBV infection, the expression level of TGF-β2 will decrease (Figure 7(b)). In addition, HepG2-NTCP       Journal of Oncology cells were transfected with knockdown CREBBP in 24 wells, transfected for 48 hours, infected with HBV at a cell density close to 100%, and then harvested 5 days after infection to measure intracellular TGF-β2. Te mRNA level is reduced (Figure 7(c)). At the same time, compared with normal liver tissues, TGF-β2 is highly expressed in liver cancer tissues associated with liver fbrosis induced by HBV infection (Figures 7(d) and 7(e)). Te above results indicate that acetylation of CREB binding protein mediates the expression of TGF-β2 induced by HBV infection.

Discussion
Te hepatitis B virus causes liver damage by involving hepatocytes, macrophages, and hematopoietic stem cells in a complex process [19,20]. Among the various cytokines related to liver fbrosis, TGF-β has been proven to be the most important [21]. Previously, it was believed that TGF-β can be secreted by hepatocytes and macrophages to activate hepatic stellate cells [22]. Increasing evidence shows that transforming growth factor-β mainly transmits signals through Smad [23]. In addition, there are reports that Smad helps β-catenin transport to the nucleus of hematopoietic stem cells and initiates the expression of fbrosis genes [24]. Te research of antifbrotic drugs is still a top priority. We found that HBV infection can promote the increase of CREBBP acetylation level and highly enrich in TGF beta pathway through acetylation-modifed proteomics detection. In recent years, etiological treatment, antiinfammatory and liver protection, inhibition of hepatic stellate cell activation and proliferation, reduction of excessive production of extracellular matrix, and acceleration of ECM degradation have become important means to inhibit the development of liver fbrosis [25]. However, there is no report on the molecular biology of acetylation-related fbrosis. Diferential proteins are strongly enriched in the "transcriptional coactivator activity" molecular functions in the cluster analysis heat map of GO classifcation and in the TGF-β signaling pathway of the KEGG pathway. Te differentially modifed proteins are enriched in the CBP/p300 atypical loop domain and CBP/p300 histone acetyltransferase domain. Finally, through cell experiments and immunohistochemistry of liver cancer tissue samples, it was verifed that HBV infection caused the increase of TGFbeta expression and the high expression of TGFbeta in liver cancer tissue. However, there is no report on the molecular   biology of acetylation-related fbrosis. Our study elucidated the new molecular mechanism of fbrosis-related liver cancer caused by HBV infection from the protein modifcation level. So far, most antifbrotic drugs are still in the preclinical research stage. In the clinical research stage, there are also some drugs with obvious antifbrotic efects, good safety, and good tolerance. It is believed that with in-depth research on the pathogenesis of liver fbrosis and the continuous advancement of new drug development, it will be possible to reverse liver fbrosis.

Data Availability
Te datasets during the current study are available from the corresponding author on reasonable request.

Disclosure
Xiuling Li and Suofeng Sun are the co-corresponding authors.

Conflicts of Interest
Te authors declares that they have no conficts of interest.