NAT10 promotes the activation of hepatic stellate cells by modulating the TGF-β1-ac4C- COL1A1  axis

NAT10 promotes the activation of hepatic stellate cells by modulating the TGF-β1-ac4C- COL1A1 axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article NAT10 promotes the activation of hepatic stellate cells by modulating the TGF-β1-ac4C- COL1A1 axis An Zhang, Jinming Shi, Fei Guan, Yuqi Zhang, Najiya Abudula, Xuemei Shao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7284137/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Hepatology International → Version 1 posted 5 You are reading this latest preprint version Abstract Background Liver fibrosis is characterized by deposition of excessive extracellular matrix (ECM). The major source of ECM is activated hepatic stellate cells (HSCs). NAT10 is the only known acetyltransferase catalyzing ac4C RNA modification. The purpose of this study is to explore the role of NAT10 acting as ac4C acetyltransferase during HSC activation. Methods NAT10 was detected in fibrotic liver tissues from S. japonicum infected mice with immunohistochemistry and TGF-β1 stimulated LX-2 human HSC cells with Western blot, immunofluorescent staining and qPCR. NAT10 was inhibited with specific siRNA in LX-2 cells to detect HSC activation molecular marker with Western blot, cell motility with Transwell assay, cell proliferation with CCK8 assay. ac4C modification was assessed in TGF-β1 stimulated LX-2 cells with immunofluorescent staining. ac4C bisulfite sequencing and transcriptomic sequencing analysis were performed to analyze ac4C modified genes regulated by NAT10 in TGF-β1 stimulated LX-2 cells. Possible target genes regulated by NAT10 were determined using qPCR, ac4C-RIP-qPCR, RNA stability assay, and were further verified using primary hepatic stellate cells from mice and using analysis with GEO datasets. Results NAT10 increases in S. japonicum infected mice liver and activated HSCs. NAT10 is correlated with TGFB1 and COL1A1 expression in activated HSCs and NAT10 inhibition suppresses HSCs activation. NAT10 promotes the ac4C modification and stability of TGFB1 and COL1A1 mRNA, thus enhancing their protein expression. Conclusions NAT10 acts as ac4C acetyltransferase and forms a positive feedback with TGF-β1 in HSCs, thus modulating the TGF-β1-ac4C- COL1A1 axis, to promote the HSCs activation and contributes to liver fibrosis. hepatic fibrosis hepatic stellate cells NAT10 N4-acetylcytosine TGF-β1 COL1A1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights 1. NAT10 catalyzes ac4C modification of and mRNA, to enhance the expression of TGF-β1 and collagen I, thus promotes the activation of hepatic stellate cells 2. NAT10 expression is correlated with and expression in activated HSCs 3. NAT10 forms a positive feedback with profibrogenic cytokine TGF-β1, to promote hepatic fibrosis Introduction Liver fibrosis is a reversible wound-healing pathological process, which follows acute or chronic liver injuries, including viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, parasitic infections like schistosomiasis and autoimmune diseases [ 1 ] . Hepatic stellate cells (HSCs) are nonparenchymal cells in the liver that play important roles in promoting the development of liver fibrosis [ 2 ] . Quiescent HSCs are lipid droplets-storing cells. Upon acute or chronic liver damage, HSCs transdifferentiate toward myofibroblast-like cells, characterized by decreased lipid droplets, increased proliferation, remodeling of cytoskeleton proteins including vimentin and α-smooth muscle actin (α-SMA), enhanced synthesis and abnormal accumulation of extracellular matrix (ECM) e.g. collagen, ultimately leading to liver fibrosis [ 2 ] , which may further develop into cirrhosis and hepatocellular carcinoma (HCC) [ 1 ] . The activation of HSCs involves multiple signaling pathways such as TGF-β/SMAD, PDGF, NK-κB [ 2 ] . Transforming growth factor-β (TGF-β) is an important inflammatory cytokine that promotes tissue fibrosis, mainly leading to tissue scar formation by activating its downstream SMAD signaling pathway [ 3 ] . TGF-β1, the most active sub-type in the TGF-β family with the highest proportion, mainly promotes nuclear entry of transcription factors including SMAD2/3, to enhance the expression of pro-fibrotic genes, thus facilitating the development of hepatic fibrosis [ 3 ] . RNA modification is an important mechanism regulating RNA metabolism and gene expression in cells to maintain cellular homeostasis, and plays a crucial regulatory role in various diseases [ 4 – 5 ] . N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), N7-methylguanosine (m7G), N4-acetylcytosine (ac4C), pseudouridine (PSI), and 2'-O-Methylation (Nm) are several common RNA modification mechanisms in cells [ 6 – 8 ] . ac4C RNA modification is the only discovered RNA acetylation modification. ac4C was reported to be mainly enriched in the coding sequence (CDS) region in human HeLa cells [ 8 ] and the main function of ac4C in mRNA modification is to enhance the stability and translation efficiency of mRNA [ 8 ] . NAT10/Kre33 is discovered to be a specific protein catalyzing ac4C RNA modification [ 9 – 10 ] . NAT10, a nucleolar localization protein containing 982 amino acids, was initially identified as an evolutionarily conserved member of the GCN5 associated N-acetyltransferase (GNAT) superfamily [ 9 – 10 ] . Structural biology studies have shown that NAT10 has typical acetyl-CoA binding sites and an RNA binding domain, which modifies various substrates including histones, non-histone proteins and RNA through acetyl transferring reactions [ 9 – 10 ] . Recent studies showed that NAT10 plays a vital role in multiple liver diseases including MASLD (metabolic dysfunction-associated steatotic liver disease), MASH (metabolic dysfunction-associated steatohepatitis) and HCC [ 11 – 12 ] . These findings collectively underscore the roles of NAT10 as ac4C acetyltransferase in liver diseases. However, the roles of NAT10 in hepatic fibrosis, especially in the activation of hepatic stellate cells, remains poorly understood. In this study, we used human HSC cell line LX-2 stimulated by TGF-β1 and in-vitro culture activated primary HSCs from mice as activated HSC cell model, to explore the possible regulatory role of ac4C mRNA modification mediated by NAT10 in hepatic stellate cells, thus to reveal the detailed mechanism of hepatic fibrosis. Methods Ethics statement and animal study. Animal experiments were approved by the Committee on Animal Research of Tongji Medical College, Huazhong University of Science and Technology, Hubei Province, PR China. Mice infection with S. japonicum cercariae was performed as previously described [ 13 ] . Isolation of primary cells, cell culture, transfection and stimulation. Isolation and cultivation of primary hepatic stellate cells from healthy BALB/c mouse were performed by in-situ digestion of the liver with collagenase IV/pronase E and Percoll density gradient centrifugation, as previously described [ 14 ] . LX-2, human HSC line, was stimulated by 5ng/ml TGF-β1 (PeproTech, USA). siRNA specific to NAT10 was transfected into cells using Lipofectamine 2000 (Invitrogen, USA). CHX (10 µg/ml, MCE, China) and ActD (4 µg/ml, MCE, China) were used to respectively inhibit the synthesis of novel protein and novel RNA in LX-2 cells in this study. Transwell (Boyden Chamber) assay. Incorporated 8-µm pore inserts (Corning, USA) were placed in a 24-well plate containing 600 µl DMEM complete medium, supplemented with 5ng/ml TGF-β1. 5 × 10 4 cells in 200 µl DMEM complete medium were added to the insert and incubated at 37°C for 24 h. The cells were fixed by 4% paraformaldehyde and counter-stained with 0.05% crystal violet solution (Biosharp, China). The non-migrating cells on the top side of the membrane were removed with a wet cotton swab. Air dried membranes were mounted and examined under microscope. The cells from 5 to 10 randomly selected fields were counted. CCK8 assay. 3×10 3 cells were seeded in each well of a 96-well microplate and treated with TGF-β1 for 24h. The culture medium was replaced with 100 µl of the corresponding culture medium containing 10% CCK8 solution (Dojindo, Japan) and incubated at 37°C for 4h. The absorbance was read at a wavelength of 450 nm in a microplate reader (Biotek, USA). Antibodies. Primary antibodies used for Western blot, immunohistochemistry and immunocytochemistry are listed as followed: ac4C (ab252215, Abcam, USA), NAT10 (13365-1-AP, Proteintech, China), α-SMA (ab32575, Abcam, USA), collagen-I (67288-1-Ig, Proteintech, China), TGF-Beta 1 (81746-RR, Proteintech), Vimentin (10366-1-AP, Proteintech), Pan-acetylation (66289-1-Ig, Proteintech), GAPDH (60004-1-lg, Proteintech, China). ac4C bisulfite sequencing. ac4C bisulfite sequencing was performed by SeqHealth Technology Co., Ltd (Wuhan, China). The integrity of total RNA extracted from LX-2 cells was confirmed by Agilent 5300 (Agilent, USA). mRNA was purified with KAPA mRNA capture kit (KK8441, Roche, USA). A small amount of purified RNA was used as "Con". The remaining RNA was incubated with 100 mM NaBH4 at 55 ℃ in the dark for 30 minutes and precipitated. The library was constructed using KC Digital strand mRNA Library Prep Kit for Illumina (DR085-02, Illumina, USA). Enrichment, quantification, and final sequencing of library products corresponding to 200–500 bps were performed using the PE150 model on MGISEQ-T7 (MGI, China). Raw reads were processed using fastp (version 0.23.0) to remove residual adaptor sequences and low-quality reads. The clean reads were mapped to the reference genome using STAR software, and duplicated reads were removed using UMI. SNV locus was detected using the pileup mode of JACUSA software and statistical tests was perform using Rigel software, to identify differential ac4C loci. Motif enrichment was performed using Home. Gene Ontology (GO) analysis of annotated genes and Kyoto Encyclopedia of Genomes (KEGG) enrichment analysis were performed using KOBAS software, with p value < 0.05 as the criterion for determining statistically significant enrichment. The Integrated Genomics Viewer (IGV) was used to track and display the distribution of ac4C modified sites. Transcriptomic sequencing and bioinformatic analysis. RNA-seq experiments were performed by Novogene (Beijing, China) and differential pathways were selected for GO, GSEA, KEGG pathway analysis (NovoMagic v3.0). All differentially expressed genes were determined by |log2FoldChange| >1 and p value < 0.05. Venny analysis was performed online at https://bioinfogp.cnb.csic.es/tools/venny/index.html . Protein functional enrichment was analyzed online at https://metascape.org/gp/index.html . qPCR and ac4C-RIP-qPCR. For ac4C-RIP, total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA) and 300 µg total RNA was incubated with 4µg anti-ac4C antibodies or IgG antibodies in 500µl IP buffer (150 mM NaCl, 0.1% NP-40, 10mMTris-HCl, pH 7.4). The mixtures were incubated with secondary antibodies conjugated with magnetic beads (Thermo, USA) and washed with IP buffer. RNA was extracted using TRIzol reagent and quantified by qPCR. For qPCR, 2 µg of RNA was reversely transcribed to cDNA with ReverTra Ace qPCR RT kit (Thermo, USA). Gene expression was quantified using Hieff qPCR SYBR Green Master Mix (Yeasen, China) on CFX Connect Real-Time system (Bio-Rad, USA). Relative expression of target gene was analyzed using established ∆∆Ct threshold method. Primers used in this study was listed in Table 1. RNA stability assay. LX-2 cells were treated with Actinomycin D at a final concentration of 4 µg/mL for the indicated time periods and collected. Total RNAs were extracted and analyzed with qPCR. β-actin was used for normalization. Western blot, immunoprecipitation, immunohistochemistry and immunocytochemistry. The performance of these experiments were carried out according to previous description [ 13 – 14 ] . Statistical analysis. All data are expressed as mean ± SD. Differences between experimental and control groups were assessed by one-way ANOVA using GraphPad Prism 10. p < 0.05 was considered statistically significant. Result 1. NAT10 expression is increased in the tissues of hepatic fibrosis and TGF-β1 stimulated LX-2 cells. Mice were infected with S. japonicum cercariae to induce liver fibrosis, with the aim to detect NAT10 expression in fibrotic liver tissues. As compared with non-infected mice, NAT10 significantly increased in the livers of S. japonicum infected mice (Fig. 1 a). Human HSC cell line, LX-2, stimulated by TGF-β1, was used as activated HSC cell model, to detect NAT10 expression. Upon TGF-β1 stimulation, NAT10 increased in LX-2 cells along with the increase of activation markers of HSCs, α-SMA (Fig. 1 b). The increased expression of NAT10 and α-SMA in TGF-β1 stimulated LX-2 cells was also detected with Immunofluorescence assay. NAT10 was mainly distributed in the nucleus, especially in the nucleolus (Fig. 1 c). Immunofluorescence assay confirmed that TGF-β1 stimulation enhanced ac4C modification abundance in LX-2 cells (Fig. 1 d). These findings collectively suggested heightened expression of NAT10 in mice fibrotic livers and TGF-β1 activated human LX-2 cells, which provide substantial evidence for a significant correlation of NAT10 mediated ac4C RNA modification with HSC activation and liver fibrosis. 2. ac4C-seq analysis indicates that TGF-β1 enhances ac4C RNA modification of LX-2 cells and differential acetylated genes are enriched in pathways of hepatic diseases. ac4C sequencing was performed using mRNA enriched from TGF-β1 stimulated LX-2 cells. Compared with control cells, the mRNA from TGF-β1 activated LX-2 cells showed the enhanced ac4C modification of mRNA (Fig. 2 a). 11746 acetylated cytosines were detected in TGF-β1 activated LX-2 cells and 8132 sites were detected in control cells (Fig. 2 b). As reported in human HeLa cells [ 8 ] , ac4C modification was also enriched in CDS (coding sequence) region of mRNAs from TGF-β1 activated LX-2 cells (Fig. 2 c and 2 d). Motifs centered around ac4C modified cytosines demonstrated the alteration of ac4C located preference sequence in LX-2 cells upon TGF-β1 stimulation (Fig. 2 e). A higher frequency of GGG upstream of the ac4C site was observed in the probability sequence context from TGF-β1 activated LX-2 cells, as compared with control cells (Fig. 2 e). Differentially acetylated genes were also subjected to KEGG pathway enrichment analysis and significant enrichment for the “Hepatocellular carcinoma”, “Hepatitis B”, “NAFLD” (Fig. 2 f) was observed. Collectively, ac4C sequencing analysis indicated the enhanced ac4C mRNA modification in TGF-β1 activated LX-2 cells. 3. NAT10 inhibition suppresses LX-2 activation upon TGF-β1 stimulation, leading to the transcriptomic alteration. NAT10 is currently the only protein discovered to catalyze ac4C RNA modification [ 10 ] , siRNA specific to NAT10 was used to inhibit NAT10 expression, to assess the role of NAT10 to LX-2 activation. The marked elevation of α-SMA was observed in LX-2 cells stimulated with TGF-β1, while NAT10 inhibition resulted in the loss of increase of α-SMA in TGF-β1 stimulated LX-2 cells (Fig. 3 a). Enhanced migration capability related with skeletal protein remodeling including vimentin and α-SMA is a typical feature of activated HSCs. The results of Transwell assay demonstrated that the enhanced migration of LX-2 cells upon TGF-β1 stimulation was suppressed by NAT10 inhibition (Fig. 3 b). Activated HSCs was also characterized with enhanced proliferation. CCK8 assay indicated that TGF-β1 stimulated LX-2 cells exhibited more increased proliferation, as compared with NAT10 siRNA transfected cells (Fig. 3 c). Transcriptomic analysis (Fig. 3 d) revealed that TGF-β1 stimulation led to the upregulation of 841 genes and the downregulation of 552 genes (Fig. 3 e), while in NAT10 siRNA transfected cells, there were 408 genes upregulated and 298 genes downregulated upon TGF-β1 stimulation (Fig. 3 f). In conclusion, the above results suggested that NAT10 inhibition suppressed TGF-β1 induced LX-2 activation, accompanied with transcriptomic alteration. 4. Transcriptomic analysis indicates that NAT10 regulates ECM-receptor interaction in TGF-β1 activated LX-2 cells. NAT10 was reported to modify various substrates including histones, non-histone proteins, and RNA through acetyl transfer reactions [ 10 ] . Co-immunoprecipitation was performed to detect whether NAT10 acts as protein acetyltransferase and enhancement of acetylation of protein complex combined with NAT10 was not detected upon TGF-β1 stimulation (Supplementary Fig. 1), which indicated that NAT10 might primarily function as an RNA acetyltransferase rather than a protein acetyltransferase in LX-2 cells. Differential genes from TGF-β1 stimulated cells (TGFB) and cells transfected with NAT10 siRNA and then stimulated with TGF-β1 (siNAT10_TGFB) were enriched and analyzed. GO (Fig. 4 a) and KEGG analysis (Fig. 4 b) indicated that differential genes of TGFB and siNAT10_TGFB are enriched in multiple pathways related with ECM function, including ECM-receptor interaction, which was confirmed by enrichment plot from Gene set enrichment analysis (GSEA) (Fig. 4 c). Venny analysis was further performed online using 841 genes upregulated in TGF-β1 activated LX-2 cells (TGFb-DEG-up), 408 genes upregulated in NAT10 siRNA transfected and further TGF-β1 stimulated LX-2 cells (siNAT10-DEG-up) enriched by transcriptomic analysis, 5402 differential genes with enhanced ac4C modification in TGF-β1 activated LX-2 cells enriched by ac4C sequencing analysis (TGFb-ac4C) (Fig. 4 d). 52 genes with enhanced ac4C modification, which was upregulated TGFb-DEG-up, however not upregulated in siNAT10-DEG-up, were regarded as candidate genes regulated by NAT10 via ac4C modification (Fig. 4 d) and input into Metascape online analysis, indicating significant gene enrichment for “Extracellular matix organization” and “ECM proteoglycans” (Fig. 4 e). Collectively, RNA-seq and ac4C-seq analysis demonstrated that target genes regulated by NAT10 via ac4C RNA modification in TGF-β1 activated LX-2 cells are enriched in ECM-receptor interaction and related pathways . 5. NAT10 regulates TGFB1 and COL1A1 expression via ac4C modification in activated LX-2 cells. As differential genes from TGFB and siNAT10_TGFB were analyzed, “TGF-beta signaling pathway” was noticed from GSEA analysis (Fig. 5 a). TGFB1 was enriched in the heatmap of “TGF-beta signaling pathway” (Supplementary Fig. 2). In the heatmap of GSEA enrichment plot “ECM-receptor interaction”, COL1A1 , encoding collagen type I, is the top ranked gene (Supplementary Fig. 2). TGFB1 and COL1A1 are among the 52 genes selected from the Venny analysis presented in Fig. 4 d, which were regarded as candidate genes regulated by NAT10 via ac4C modification. TGF-β1, an important inflammatory cytokine promoting tissue fibrosis, was used in this study to activate LX-2 cells. We therefore suppose that TGF-β1 enhances NAT10 expression and NAT10 catalyzes ac4C modification of TGFB1 mRNA, to form a positive feedback, promoting ac4C modification and expression of COL1A1 mRNA. Transcriptional expression of TGFB1 and COL1A1 , was detected by qPCR, which indicated that the increased mRNA levels of TGFB1 and COL1A1 in TGF-β1 activated LX-2 cells (Fig. 5 b), was suppressed by NAT10 inhibition (Fig. 5 b). The Integrated Genomics Viewer (IGV) was used to track and display the distribution of ac4C modified sites detected by ac4C-sequencing. Compared with the control cells, COL1A1 and TGFB1 mRNA from the TGF-β1 stimulated cells showed more sites with ac4C modification (Supplementary Fig. 3). To verify the ac4C sequencing results, ac4C-RIP-qPCR was performed to determine the enhanced ac4C modification of TGFB1 and COL1A1 mRNA in TGF-β1 stimulated LX-2 cells (Fig. 5 c). ac4C modification of TGFB1 and COL1A1 mRNA was also detected in NAT10 siRNA transfected cells, however, the results indicated “not detectable” due to exceeding the detection range (data not shown). The effect of NAT10 to TGFB1 and COL1A1 mRNA stability was evaluated by RNA stability assay (Fig. 5 d-e). Stability of both COL1A1 (Fig. 5 d) and TGFB1 (Fig. 5 e) mRNA was enhanced in LX-2 upon TGF-β1 stimulation, while enhanced mRNA stability of these genes was abolished by NAT10 inhibition (Fig. 5 d-e). Expression of collagen I and TGFB1 at protein level was further detected and the results were consistent with mRNA level (Fig. 5 f). Overall, the above results indicated that in TGF-β1 activated LX-2 cells, NAT10 regulated transcriptional and translational expression of TGFB1 and COL1A1, via ac4C modification. 6. NAT10 expression is increased in activated primary HSCs and NAT10 correlates with TGFB1 and COL1A1 expression. In order to verify the results from human LX-2 cells, primary HSCs were isolated from mice liver and cultured to naturally activate in-vitro . As primary HSCs were cultivated from 2 to 6 days in-vitro , the expression of HSC activation marker molecules α-SMA and vimentin increased, indicating cell activation (Fig. 6 a). Along with cell activation, the expression of NAT10, TGFB1, and collagen I also increased (Fig. 6 a). NAT10 siRNA was transfected into primary HSCs from mice at 2 days after isolation and seeding, to detect Tgfb1 and Col1a1 at mRNA level. In primary HSCs from mice, mRNA expression of Nat10 , Tgfb1 and Col1a1 in cells cultured to 6 days was enhanced, as compared with cells cultured to 2 days (Fig. 6 b), while in Nat10 inhibited cells, mRNA expression of Tgfb1 and Col1a1 was inhibited, as compared with non-specific siRNA transfected cells (Fig. 6 b). NASH (non-alcoholic steatohepatitis) is currently the main cause of liver fibrosis in humans. Activation of HSCs is an important mechanism driving the progression of NASH to liver fibrosis [ 15 ] . A Gene Expression Omnibus (GEO) dataset [ 16 ] related to human NASH, GSE212837, has been selected to evaluate the expression of NAT10 , TGFB1 and COL1A1 , and the correlation between these genes. GSE212837 is a single-cell sequencing dataset. According to the marker genes described [ 16 ] , HSC cell population was separated to analyze the expression of NAT10 , TGFB1 and COL1A1 using R language ( http://www.R-project.org/ ). HSCs of NASH group displayed an increased expression of NAT10 , TGFB1 and COL1A1 , as compared with control group (Fig. 6 c). Linear regression analysis using R showed a strong correlation between NAT10 and TGFB1 (R 2 = 1.000), and a moderate correlation between NAT10 and COL1A1 (R 2 = 0.425) in the NASH group (Fig. 6 d). In the Human Liver Proteome Database ( http://www.liverproteome.org/ ), TGFB1 and COL1A1 were also retrieved as significantly expressed proteins in liver cirrhosis (Fig. 6 e). To be summarized, the above results based on in-vitro activated primary HSCs from mice and human NASH GEO dataset analysis indicate that NAT10 expression is increased in activated HSCs and NAT10 correlates with TGFB1 and COL1A1 expression. Discussion In current study, TGF-β1 activated human LX-2 cells and in-vitro activated primary hepatic stellate cells from mice were used as activated HSC cell model, to explore the role of NAT10 mediated ac4C RNA modification in the activation of HSCs. NAT10 catalyzes the acetylation of various substrates including proteins and RNA. Previous studies have determined that NAT10 acts as a lysine acetyltransferase to acetylate α-tubulin, p53 and histones [ 17 – 20 ] . This study did not clarify the increased lysine acetyltransferase activity of NAT10 in LX-2 cells stimulated by TGF-β1. However, the enzyme's activity is highly specific, and this result does not rule out the possibility that NAT10 may act as lysine acetyltransferase to catalyze acetylation of certain specific proteins. Further experiments are essential to clarify the activity of lysine acetyltransferase of NAT10 during HSCs activation. In this study, the enhanced expression of NAT10 was determined in liver fibrosis tissue from schistosomiasis japonicum infected mice and LX-2 human HSCs activated by TGF-β1. In in-vitro activated primary HSCs from mice, NAT10 was defined to increase at both mRNA level and protein level. Analysis of GSE212837 from a study on human NASH [ 16 ] also revealed enhanced expression of NAT10. Recent studies have reported the mechanism by which NAT10 expression is regulated [ 21 – 23 ] . At the transcriptional level, it was reported that transcriptional factor Hif-1 regulates NAT10 expression in gastric cancer cells [ 22 ] . We have previously reported that Hif−1 promotes HSC activation via regulating its multiple target genes [ 13 – 14 , 24 ] . Potential transcription factors regulating NAT10 expression were analyzed through the JASPER transcription factor online analysis, which suggested that multiple transcription factors including Hif−1, NRF1, ZNF460 and ZNF284, may regulate NAT10 expression (supplementary Fig. 4). In addition to transcriptional regulation, post-translational modification enhances stability of NAT10 at protein level. It was reported that Khib modification (2-hydroxyisobutyrylation) of NAT10 enhances the interaction of NAT10 with deubiquitinase USP39, resulting in increased NAT10 protein stability [ 23 ] . The mechanism by which NAT10 increases in activated hepatic stellate cells deserves further exploration. TGF-β1/Smad signaling pathway is an important pathway that promotes the HSCs activation [ 3 ] . Analysis of GSE212837 from a study on human NASH [ 16 ] revealed a strong correlation between NAT10 and TGFB1 in activated HSCs in NASH. Enhanced expression of NAT10 leads to increased ac4C mRNA modification in LX-2 cells, including enhanced ac4C modification of TGFB1 mRNA, leading to increased RNA stability of TGFB1 , and subsequently sustained enhancement of TGF-β1 protein expression. These results suggested that TGF-β1 and NAT10 form positive feedback through the ac4C modification in activated HSCs. Target genes regulated by NAT10 are enriched in ECM-receptor interaction in TGF-β1 stimulated LX-2 cells. The top gene enriched in “ECM-receptor interaction” is COL1A1 , encoding collagen I. Results from LX-2 cells and primary HSCs from mice, verified that COL1A1 is target gene regulated by NAT10 via ac4C modification. Recent reports have revealed that NAT10 inhibition suppresses the progression of multiple liver diseases [ 11 , 25 ] . In current study, the results revealed that NAT10 acts as ac4C acetyltransferase and forms a positive feedback with TGF-β1 in HSCs, thus modulating the ac4C modification of TGFB1 and COL1A1 mRNA, which improves the stability of TGFB1 and COL1A1 mRNA, promotes protein expression of TGF-β1 and collagen I, facilitating HSCs activation and the progression of liver fibrosis (Fig. 6 f). Targeting NAT10 regulated TGF-β1-ac4C- COL1A1 axis might be a promising direction for intervening in liver fibrosis. Abbreviations .ac4C (N4-acetylcytosine); α-SMA (α-smooth muscle actin); CDS (coding sequence); CHX (cycloheximide); COL1A1 (collagen type I alpha 1 chain); ECM (extracellular matrix); HCC (hepatocellular carcinoma); HSC (hepatic stellate cell); MASH (metabolic dysfunction-associated steatohepatitis); MASLD (metabolic dysfunction-associated steatotic liver disease); MMPs (matrix metalloproteinases); NASH (non-alcoholic steatohepatitis), NAT10 (N-acetyltransferase 10); NK-κB (nuclear factor kappa B subunit); PDGF (platelet-derived growth factor); TIMPs (tissue metalloproteinase inhibitors); TGF-β1 (transforming growth factor-beta1); TGFB1 (transforming growth factor-beta1) Declarations Disclosure Statement No conflict of interest exists. Author contributions: AZ and CS conceived and designed the project. AZ performed most of the experiments. FG, YZ, JS, NA, XS, QQ, WL performed some of the experiments. AZ, JS, NA and CS analyzed and interpreted the data. AZ and CS wrote the manuscript. All the authors have read this manuscript. Financial support This work was supported by National Natural Science Foundation of China (No.c, to Shi C), Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, Huazhong University of Science and Technology (No.500153003). Acknowledgement We sincerely thank Dr. Lu Wan (Department of Pathophysiology, School of Basic Medicine, Huazhong University of Science and Technology) for kindly reviewing the manuscript. We thank Dr. Yuyu Xie (Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology) and Dr. Zhangbo Cui (School of Public Health, Tongji Medical College, Huazhong University of Science and Technology) for the guidance and assistance of animal study. References Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis. Lancet. 2014;383(9930):1749-1761. Higashi T, Friedman SL, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev. 2017;121:27-42. 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Multi-modal analysis of human hepatic stellate cells identifies novel therapeutic targets for metabolic dysfunction-associated steatotic liver disease. J Hepatol. 2025;82(5):882-897. Wang S, Li K, Pickholz E, et al. An autocrine signaling circuit in hepatic stellate cells underlies advanced fibrosis in nonalcoholic steatohepatitis. Sci Transl Med. 2023;15(677):eadd3949. Jin C, Wang T, Zhang D, et al. Acetyltransferase NAT10 regulates the Wnt/β-catenin signaling pathway to promote colorectal cancer progression via ac4C acetylation of KIF23 mRNA. J Exp Clin Cancer Res. 2022;41(1):345. Larrieu D, Britton S, Demir M, et al. Chemical inhibition of NAT10 corrects defects of laminopathic cells. Science. 2014;344(6183):527-532. Larrieu D, Viré E, Robson S, et al. Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway. Sci Signal. 2018;11(537):eaar5401. Liu X, Tan Y, Zhang C, et al. NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2. EMBO Rep. 2016;17(3):349-366. Lv J, Liu H, Wang Q, et al. Molecular cloning of a novel human gene encoding histone acetyltransferase-like protein involved in transcriptional activation of hTERT. Biochem Biophys Res Commun. 2003;311(2):506-513. Zhang G, Zheng B, Chen X, et al. N4-Acetylcytidine Drives Glycolysis Addiction in Gastric Cancer via NAT10/SEPT9/HIF-1α Positive Feedback Loop. Adv Sci (Weinh). 2023;10(23):e2300898. Liao L, He Y, Li SJ, et al. Lysine 2-hydroxyisobutyrylation of NAT10 promotes cancer metastasis in an ac4C-dependent manner. Cell Res. 2023;33(5):355-371. Liu J, Xie Y, Cui Z, et al. Bnip3 interacts with vimentin, an intermediate filament protein, and regulates autophagy of hepatic stellate cells. Aging (Albany NY). 2020 Dec 3;13(1):957-972. Liu H, Xu L, Yue S, et al. Targeting N4-acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2-mediated HMGB2 mRNA translation. Cancer Commun (Lond). 2024;44(9):1018-1041. Table 1 Table 1 is available in the Supplementary Files section. Supplementary Files table1.xlsx supplementaryfigure1NAT10aceIP.tif Supplementary figure 1. LX-2 cells were stimulated with 5ng/ml TGF-β1 and cell lysates were collected. Co-immunoprecipitation was performed to detect pan-acetylation of protein complex combined with NAT10. Pan-Ace: pan-acetylation. supplementaryfigure2.tif Supplementary figure 2. Heatmap of GSEA enrichment plot “ECM-receptor interaction” and “TGF-beta signaling pathway” from control cells and TGF-β1 stimulated LX-2 cells. supplementaryfigure3IGV.tif Supplementary figure 3. The Integrated Genomics Viewer (IGV) was used to track and display the distribution of ac4C modified sites detected by ac4C-sequencing in COL1A1 mRNA and TGFB1 mRNA in TGF-β1 stimulated LX-2 cells and control cells. supplementaryfigure4.tif Supplementary figure 4. Online prediction of transcription factors possibly associated with NAT10 expression using JASPER ( href="http://jaspar.genereg.net/%EF%BC%89%EF%BC%8C%E6%8C%89%E7%89%A9%E7%A7%8D%E3%80%81%E5%AE%B6%E6%97%8F%E6%88%96%E5%9F%BA%E5%9B%A0%E6%90%9C%E7%B4%A2%EF%BC%88%E5%A6%82%E8%BE%93%E5%85%A5%E2%80%9CMYC%E2%80%9D%EF%BC%89%E3%80%82">http://jaspar.genereg.net/). Cite Share Download PDF Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Hepatology International → Version 1 posted Editorial decision: Major Revisions Needed 12 Sep, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers invited by journal 17 Aug, 2025 Editor assigned by journal 06 Aug, 2025 First submitted to journal 05 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7284137","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501423128,"identity":"ab4b0bb7-b35a-4c69-90a4-b51df2077b96","order_by":0,"name":"An Zhang","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"An","middleName":"","lastName":"Zhang","suffix":""},{"id":501423129,"identity":"bcb3551a-2bc0-4282-a1ff-9c16c83164df","order_by":1,"name":"Jinming Shi","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jinming","middleName":"","lastName":"Shi","suffix":""},{"id":501423130,"identity":"e56b3ad5-e83e-4642-a005-a2e726e6cffa","order_by":2,"name":"Fei Guan","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Guan","suffix":""},{"id":501423131,"identity":"54069380-d7dc-4d10-9a16-a30e6b2450ed","order_by":3,"name":"Yuqi Zhang","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yuqi","middleName":"","lastName":"Zhang","suffix":""},{"id":501423132,"identity":"8c1799a6-6813-4f6e-9fe4-1f1ef78c5c59","order_by":4,"name":"Najiya Abudula","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Najiya","middleName":"","lastName":"Abudula","suffix":""},{"id":501423133,"identity":"ecc98dba-f24b-4a10-a25f-7e158cbcc5dc","order_by":5,"name":"Xuemei Shao","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xuemei","middleName":"","lastName":"Shao","suffix":""},{"id":501423134,"identity":"53fa2646-dd9c-4fa6-9cf9-6f9046e2a51c","order_by":6,"name":"Qianwei Qi","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Qianwei","middleName":"","lastName":"Qi","suffix":""},{"id":501423135,"identity":"61578701-d002-4ff4-ba27-b43215e67882","order_by":7,"name":"Wentao Liu","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Wentao","middleName":"","lastName":"Liu","suffix":""},{"id":501423136,"identity":"85420fe2-d0cd-4f85-ac60-0fdd877f1cb7","order_by":8,"name":"Chunwei Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACCShpwMx8DCqUQLQWtjQgw4BoLSC1PGbEaeGf3WP2mKfCwt6cnefbY54/fxj42XMMGH7uwGPJnTPmxjxnJBJ3NvNuN+ZtM2CQ7HljwNh7BrcWA4kcM+ncNokEg8O826R5GwwYDG7kGDAzthHS8k/C3uAwzzNpnj8GDPbEaWmQYNxwmIdNmofNACSCX4vEjbQy6T/HJBI3HGYzk5zbZswjceZZwcFePFr4ZyRvk5xRU2dvcP7wM4k3f+Tk+NuTNz74iUcLBuABEQdI0DAKRsEoGAWjAAsAAC5+ROaKNlGbAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6908-4842","institution":"School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Chunwei","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-08-03 15:04:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7284137/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7284137/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12072-025-10998-x","type":"published","date":"2026-01-20T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89808388,"identity":"85e11d05-dcf0-4d14-80ac-68db1d9ff13c","added_by":"auto","created_at":"2025-08-25 09:34:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5067169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAT10 increases in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. japonicum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infected mice liver and TGF-β1 activated LX-2 hepatic stellate cells. \u003c/strong\u003e(a) BALB/c female mice, 6–8 weeks old, were percutaneously infected with 25 cercariae of \u003cem\u003eSchistosoma japonicum\u003c/em\u003e through the shaved abdomen, sacrificed at 6 and 12 weeks post-infection, and samples of liver were collected to detect NAT10 in \u003cem\u003eS. japonicum\u003c/em\u003e-infected (n = 3) and non-infected (n = 3) mice liver with immunohistochemistry. Representative images were shown (Scale bar, 50 μm). Positive cells were counted and the scores was multiplied with staining intensity, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (b) LX-2 cells were stimulated with 5ng/ml TGF-β1 and cell lysates were collected at indicated time to detect NAT10 with Western blot. Densitometric analysis for Western blot was performed and data were expressed as mean ± SD, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (c) Immunofluorescence assay was performed to detect NAT10 (Cy3) and α-SMA (FITC) in TGF-β1 stimulated LX-2 cells (Scale bar, 10 μm). Images were captured by confocal microscope.\u003cstrong\u003e \u003c/strong\u003e(d) Immunofluorescence assay was performed to detect ac4C (Cy3) in TGF-β1 stimulated LX-2 cells (Scale bar, 10 μm). Images were captured by confocal microscope.\u003c/p\u003e","description":"","filename":"figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/4a43367645e3eeec01a7171a.jpg"},{"id":89809435,"identity":"06184325-dd88-4777-a55a-bf6f4103d9e9","added_by":"auto","created_at":"2025-08-25 09:42:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1400433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eac4C-seq analysis indicates that TGF-β1 enhances ac4C RNA modification of LX-2 cells and differential genes with ac4C modification are enriched in pathways related with hepatic diseases.\u003c/strong\u003e LX-2 cells were stimulated with 5ng/ml TGF-β1 for 24h. Total RNA was extracted, mRNA was enriched with polyA and then sequenced by ac4C-seq chemical method. Sample were prepared from 3 biological replicates. (a) Violin diagram of ac4C modified cytosine bases. (b) Number of ac4C modified sites in control cells and TGF-β1 stimulated cells. (c) The distribution density of ac4C modified sites annotated in various elements of the transcripts. (d) The distribution of ac4C modified sites annotated in different functional regions of the genes. (e) The sequence context of 3 bases upstream and downstream of ac4C sites is depicted in the probability pattern. (f) KEGG analysis of ac4C-modified differential genes.\u003c/p\u003e","description":"","filename":"figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/a59292e6161bc29aef509590.jpg"},{"id":89809432,"identity":"ad55355c-537e-4310-b099-cfa9fd95b2cd","added_by":"auto","created_at":"2025-08-25 09:42:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2107582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAT10 inhibition suppresses the activation of LX-2 upon TGF-β1 stimulation, leading to the transcriptomic alteration of LX-2 cells. \u003c/strong\u003eLX-2 cells were transfected with specific siRNA targeting \u003cem\u003eNAT10\u003c/em\u003e and cells were stimulated with TGF-β1 24h post transfection. (a) Cell lysates were subjected t o detect NAT10and α-SMA with Western blot.Densitometric analysis was performed and data were expressed as mean ± SD, ns: not significant, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (b) Transwell assay was performed to detect cell motility. 8 fields under microscope were randomly selected for counting, and statistical analysis was performed. ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u0026nbsp;(c) CCK8 assay was performed to detect the proliferation of LX-2 cells. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ns: not significant. (d-f) Total RNA was extracted and transcriptomic sequencing analysis were performed. Sample were prepared from 3 biological replicates. (d) Cluster analysis of differentially expressed genes. (e) Volcano plot of differentially expressed genes in control group, NC: negative control LX-2 cells, TGFB: TGF-β1 stimulated LX-2 cells. (f) Volcano plot of differentially expressed genes in siRNA group, siNAT10: LX-2 cells transfected with siRNA targeting \u003cem\u003eNAT10, \u003c/em\u003esiNAT10_TGFB: LX-2 cells transfected with siRNA targeting \u003cem\u003eNAT10 \u003c/em\u003eand stimulated by TGF-β1.\u003c/p\u003e","description":"","filename":"figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/d0f6190dfe73eb0b5e94636a.jpg"},{"id":89808394,"identity":"e2527841-da7d-49b9-b63d-d16ce6468ba7","added_by":"auto","created_at":"2025-08-25 09:34:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2450291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis indicates that NAT10 regulates ECM-receptor interaction and related pathways in TGF-β1 activated LX-2 cells. \u003c/strong\u003e(a) GO analysis of transcriptomic sequencing analysis. (b) KEGG analysis of transcriptomic sequencing analysis. (c) GSEA analysis of transcriptomic sequencing analysis, enrichment plot of ECM-receptor interaction. (d) Venny analysis with differentially expressed genes (DEGs) upregulated in TGF-β1 stimulated LX-2 cells (TGF-β DEG up), DEGs with ac4C modification in TGF-β stimulated LX-2 cells (TGF-β ac4C), DEGs upregulated in LX-2 cells transfected with siRNA targeting \u003cem\u003eNAT10 \u003c/em\u003eand stimulated by (siNAT10/TGF-β DEG up). (e) 52 genes with ac4C modification and regulated by NAT10 was screened and input into metascape online analysis.\u003c/p\u003e","description":"","filename":"figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/8be2e75e61cd510ab8290a0e.jpg"},{"id":89808395,"identity":"2c34f7bc-18c6-474f-b7dc-07abaabb2f95","added_by":"auto","created_at":"2025-08-25 09:34:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1559667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAT10 promotes the ac4C modification and stability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTGFB1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCOL1A1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emRNA, thus enhancing their protein expression. \u003c/strong\u003e(a) GSEA analysis of transcriptomic sequencing analysis, enrichment plot of TGF-β signaling pathway. LX-2 cells were transfected with specific siRNA targeting \u003cem\u003eNAT10\u003c/em\u003e and cells were stimulated with TGF-β1 24h post transfection. (b) Total RNA was extracted and reversely transcribed into cDNA to detect the expression of \u003cem\u003eNAT10\u003c/em\u003e, \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e at mRNA level by qPCR. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). (c) Total RNA was extracted and ac4C-RIP-qPCR was performed to detect \u003cem\u003eTGFB1 \u003c/em\u003eand \u003cem\u003eCOL1A1\u003c/em\u003e. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). (d-e) Cells were additionally treated with Actinomycin D (Act D) for 0h, 2h, 4h, 6h, 8h and 10h. RNA was extracted to detect mRNA of \u003cem\u003eTGFB1\u003c/em\u003e(d) and \u003cem\u003eCOL1A1 \u003c/em\u003e(e) by qPCR. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). (f). LX-2 cells were transfected with specific siRNA targeting \u003cem\u003eNAT10\u003c/em\u003e and cells were stimulated with TGF-β1 24h post transfection. Cell lysates were subjected to detect TGFB1and collagen I with Western blot.Densitometric analysis was performed and data were expressed as mean ± SD, ns: not significant, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/af021f293460958304da1812.jpg"},{"id":89808397,"identity":"774eda57-1c98-425f-ae7a-f7223ff66a87","added_by":"auto","created_at":"2025-08-25 09:34:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1517798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAT10 expression is increased in activated primary hepatic stellate cells and NAT10 correlates with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTGFB1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e COL1A1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression. \u003c/strong\u003e(a) Culture-activated primary HSCs from mice were cultured up to 2 days, 4 days or 6 days. Western blot was performed to detect NAT10, HSC activation markers vimentin and α-SMA, TGFB1 and collagen I. (b) Primary HSCs were isolated from mice and cells were transfected with specific siRNA targeting NAT10 as cells were cultivated up to day 2 and cells were collected to extract total RNA. Transcriptional expression of \u003cem\u003eNat10\u003c/em\u003e, \u003cem\u003eTgfb1\u003c/em\u003e and\u003cem\u003e Col1a1\u003c/em\u003e was detected with qPCR. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01,*** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001. (c) Expression of NAT10, TGFB1 and COL1A1 in HSCs in control group and NASH group from GSE212837 database was analyzed using R. (d) Correlation analysis between \u003cem\u003eNAT10\u003c/em\u003e and\u003cem\u003e TGFB1\u003c/em\u003e, \u003cem\u003eNAT10\u003c/em\u003e and \u003cem\u003eCOL1A1 \u003c/em\u003ewas conducted through linear regression analysis using R. (e) Expression of TGFB1 and COL1A1 in cirrhosis retrieved from the Human Liver Proteome Database. (f) Schematic model of the role of NAT10 in the activation of hepatic stellate cells by modulating the TGF-β1-ac4C-\u003cem\u003eCOL1A1\u003c/em\u003e axis.\u003c/p\u003e","description":"","filename":"figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/cfe4ddb6668b61c8d7c94cc5.jpg"},{"id":101151732,"identity":"2d759b95-137a-4a95-8e89-627658ce96be","added_by":"auto","created_at":"2026-01-26 16:03:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15659313,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/a80d25e9-91be-48ff-b633-5fd7a30e5ce4.pdf"},{"id":89811060,"identity":"e94cd5e9-3318-40ec-af4e-a04cf92980a9","added_by":"auto","created_at":"2025-08-25 09:58:47","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11029,"visible":true,"origin":"","legend":"","description":"","filename":"table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/5848a3159c09d668d9079104.xlsx"},{"id":89808390,"identity":"e19d0260-af64-491b-92d0-3be08649772c","added_by":"auto","created_at":"2025-08-25 09:34:47","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":936828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 1. \u003c/strong\u003eLX-2 cells were stimulated with 5ng/ml TGF-β1 and cell lysates were collected. Co-immunoprecipitation was performed to detect pan-acetylation of protein complex combined with NAT10. Pan-Ace: pan-acetylation.\u003c/p\u003e","description":"","filename":"supplementaryfigure1NAT10aceIP.tif","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/19bfde14ac5caef4dd478784.tif"},{"id":89809439,"identity":"d9c97be0-5a7e-4f2d-a96b-3cfb81c7f9a6","added_by":"auto","created_at":"2025-08-25 09:42:47","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8230944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 2\u003c/strong\u003e. Heatmap of GSEA enrichment plot “ECM-receptor interaction” and “TGF-beta signaling pathway” from control cells and TGF-β1 stimulated LX-2 cells.\u003c/p\u003e","description":"","filename":"supplementaryfigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/1eb7b7c2287a24f3edc85780.tif"},{"id":89809436,"identity":"eb74b89c-ad45-49e0-9674-c9c4b5506d71","added_by":"auto","created_at":"2025-08-25 09:42:47","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1430852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 3. \u003c/strong\u003eThe Integrated Genomics Viewer (IGV) was used to track and display the distribution of ac4C modified sites detected by ac4C-sequencing in \u003cem\u003eCOL1A1 \u003c/em\u003emRNA and \u003cem\u003eTGFB1\u003c/em\u003e mRNA in TGF-β1 stimulated LX-2 cells and control cells.\u003c/p\u003e","description":"","filename":"supplementaryfigure3IGV.tif","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/6439fb0a2d3d48e65b4c419f.tif"},{"id":89809440,"identity":"751adb62-6cf8-4e78-b50e-aaa64c2a7557","added_by":"auto","created_at":"2025-08-25 09:42:47","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1506728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 4. \u003c/strong\u003eOnline prediction of transcription factors possibly associated with NAT10 expression using JASPER (\u003ca href=\"http://jaspar.genereg.net/%EF%BC%89%EF%BC%8C%E6%8C%89%E7%89%A9%E7%A7%8D%E3%80%81%E5%AE%B6%E6%97%8F%E6%88%96%E5%9F%BA%E5%9B%A0%E6%90%9C%E7%B4%A2%EF%BC%88%E5%A6%82%E8%BE%93%E5%85%A5%E2%80%9CMYC%E2%80%9D%EF%BC%89%E3%80%82\"\u003ehttp://jaspar.genereg.net/\u003c/a\u003e).\u003c/p\u003e","description":"","filename":"supplementaryfigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7284137/v1/11538a42ee1e7a83197a949c.tif"}],"financialInterests":"","formattedTitle":"NAT10 promotes the activation of hepatic stellate cells by modulating the TGF-β1-ac4C- COL1A1 axis","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. NAT10 catalyzes ac4C modification of and mRNA, to enhance the expression of TGF-β1 and collagen I, thus promotes the activation of hepatic stellate cells\u003c/p\u003e\u003cp\u003e2. NAT10 expression is correlated with and expression in activated HSCs\u003c/p\u003e\u003cp\u003e3. NAT10 forms a positive feedback with profibrogenic cytokine TGF-β1, to promote hepatic fibrosis\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eLiver fibrosis is a reversible wound-healing pathological process, which follows acute or chronic liver injuries, including viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, parasitic infections like schistosomiasis and autoimmune diseases\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Hepatic stellate cells (HSCs) are nonparenchymal cells in the liver that play important roles in promoting the development of liver fibrosis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Quiescent HSCs are lipid droplets-storing cells. Upon acute or chronic liver damage, HSCs transdifferentiate toward myofibroblast-like cells, characterized by decreased lipid droplets, increased proliferation, remodeling of cytoskeleton proteins including vimentin and α-smooth muscle actin (α-SMA), enhanced synthesis and abnormal accumulation of extracellular matrix (ECM) e.g. collagen, ultimately leading to liver fibrosis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, which may further develop into cirrhosis and hepatocellular carcinoma (HCC)\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe activation of HSCs involves multiple signaling pathways such as TGF-β/SMAD, PDGF, NK-κB\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Transforming growth factor-β (TGF-β) is an important inflammatory cytokine that promotes tissue fibrosis, mainly leading to tissue scar formation by activating its downstream SMAD signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. TGF-β1, the most active sub-type in the TGF-β family with the highest proportion, mainly promotes nuclear entry of transcription factors including SMAD2/3, to enhance the expression of pro-fibrotic genes, thus facilitating the development of hepatic fibrosis \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRNA modification is an important mechanism regulating RNA metabolism and gene expression in cells to maintain cellular homeostasis, and plays a crucial regulatory role in various diseases\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e–\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), N7-methylguanosine (m7G), N4-acetylcytosine (ac4C), pseudouridine (PSI), and 2'-O-Methylation (Nm) are several common RNA modification mechanisms in cells\u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e–\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. ac4C RNA modification is the only discovered RNA acetylation modification. ac4C was reported to be mainly enriched in the coding sequence (CDS) region in human HeLa cells\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e and the main function of ac4C in mRNA modification is to enhance the stability and translation efficiency of mRNA\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. NAT10/Kre33 is discovered to be a specific protein catalyzing ac4C RNA modification\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. NAT10, a nucleolar localization protein containing 982 amino acids, was initially identified as an evolutionarily conserved member of the GCN5 associated N-acetyltransferase (GNAT) superfamily\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Structural biology studies have shown that NAT10 has typical acetyl-CoA binding sites and an RNA binding domain, which modifies various substrates including histones, non-histone proteins and RNA through acetyl transferring reactions\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecent studies showed that NAT10 plays a vital role in multiple liver diseases including MASLD (metabolic dysfunction-associated steatotic liver disease), MASH (metabolic dysfunction-associated steatohepatitis) and HCC\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e–\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. These findings collectively underscore the roles of NAT10 as ac4C acetyltransferase in liver diseases. However, the roles of NAT10 in hepatic fibrosis, especially in the activation of hepatic stellate cells, remains poorly understood. In this study, we used human HSC cell line LX-2 stimulated by TGF-β1 and \u003cem\u003ein-vitro\u003c/em\u003e culture activated primary HSCs from mice as activated HSC cell model, to explore the possible regulatory role of ac4C mRNA modification mediated by NAT10 in hepatic stellate cells, thus to reveal the detailed mechanism of hepatic fibrosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eEthics statement and animal study.\u003c/b\u003e Animal experiments were approved by the Committee on Animal Research of Tongji Medical College, Huazhong University of Science and Technology, Hubei Province, PR China. Mice infection with \u003cem\u003eS. japonicum\u003c/em\u003e cercariae was performed as previously described\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation of primary cells, cell culture, transfection and stimulation.\u003c/b\u003e Isolation and cultivation of primary hepatic stellate cells from healthy BALB/c mouse were performed by \u003cem\u003ein-situ\u003c/em\u003e digestion of the liver with collagenase IV/pronase E and Percoll density gradient centrifugation, as previously described\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. LX-2, human HSC line, was stimulated by 5ng/ml TGF-β1 (PeproTech, USA). siRNA specific to NAT10 was transfected into cells using Lipofectamine 2000 (Invitrogen, USA). CHX (10 µg/ml, MCE, China) and ActD (4 µg/ml, MCE, China) were used to respectively inhibit the synthesis of novel protein and novel RNA in LX-2 cells in this study.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranswell (Boyden Chamber) assay.\u003c/b\u003e Incorporated 8-µm pore inserts (Corning, USA) were placed in a 24-well plate containing 600 µl DMEM complete medium, supplemented with 5ng/ml TGF-β1. 5 × 10\u003csup\u003e4\u003c/sup\u003e cells in 200 µl DMEM complete medium were added to the insert and incubated at 37°C for 24 h. The cells were fixed by 4% paraformaldehyde and counter-stained with 0.05% crystal violet solution (Biosharp, China). The non-migrating cells on the top side of the membrane were removed with a wet cotton swab. Air dried membranes were mounted and examined under microscope. The cells from 5 to 10 randomly selected fields were counted.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCCK8 assay.\u003c/b\u003e 3×10\u003csup\u003e3\u003c/sup\u003e cells were seeded in each well of a 96-well microplate and treated with TGF-β1 for 24h. The culture medium was replaced with 100 µl of the corresponding culture medium containing 10% CCK8 solution (Dojindo, Japan) and incubated at 37°C for 4h. The absorbance was read at a wavelength of 450 nm in a microplate reader (Biotek, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibodies.\u003c/b\u003e Primary antibodies used for Western blot, immunohistochemistry and immunocytochemistry are listed as followed: ac4C (ab252215, Abcam, USA), NAT10 (13365-1-AP, Proteintech, China), α-SMA (ab32575, Abcam, USA), collagen-I (67288-1-Ig, Proteintech, China), TGF-Beta 1 (81746-RR, Proteintech), Vimentin (10366-1-AP, Proteintech), Pan-acetylation (66289-1-Ig, Proteintech), GAPDH (60004-1-lg, Proteintech, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003eac4C bisulfite sequencing.\u003c/b\u003e ac4C bisulfite sequencing was performed by SeqHealth Technology Co., Ltd (Wuhan, China). The integrity of total RNA extracted from LX-2 cells was confirmed by Agilent 5300 (Agilent, USA). mRNA was purified with KAPA mRNA capture kit (KK8441, Roche, USA). A small amount of purified RNA was used as \"Con\". The remaining RNA was incubated with 100 mM NaBH4 at 55 ℃ in the dark for 30 minutes and precipitated. The library was constructed using KC Digital strand mRNA Library Prep Kit for Illumina (DR085-02, Illumina, USA). Enrichment, quantification, and final sequencing of library products corresponding to 200–500 bps were performed using the PE150 model on MGISEQ-T7 (MGI, China). Raw reads were processed using fastp (version 0.23.0) to remove residual adaptor sequences and low-quality reads. The clean reads were mapped to the reference genome using STAR software, and duplicated reads were removed using UMI. SNV locus was detected using the pileup mode of JACUSA software and statistical tests was perform using Rigel software, to identify differential ac4C loci. Motif enrichment was performed using Home. Gene Ontology (GO) analysis of annotated genes and Kyoto Encyclopedia of Genomes (KEGG) enrichment analysis were performed using KOBAS software, with \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 as the criterion for determining statistically significant enrichment. The Integrated Genomics Viewer (IGV) was used to track and display the distribution of ac4C modified sites.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptomic sequencing and bioinformatic analysis.\u003c/b\u003e RNA-seq experiments were performed by Novogene (Beijing, China) and differential pathways were selected for GO, GSEA, KEGG pathway analysis (NovoMagic v3.0). All differentially expressed genes were determined by |log2FoldChange| \u0026gt;1 and \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05. Venny analysis was performed online at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/index.html\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Protein functional enrichment was analyzed online at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/gp/index.html\u003c/span\u003e\u003cspan address=\"https://metascape.org/gp/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eqPCR and ac4C-RIP-qPCR.\u003c/b\u003e For ac4C-RIP, total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA) and 300 µg total RNA was incubated with 4µg anti-ac4C antibodies or IgG antibodies in 500µl IP buffer (150 mM NaCl, 0.1% NP-40, 10mMTris-HCl, pH 7.4). The mixtures were incubated with secondary antibodies conjugated with magnetic beads (Thermo, USA) and washed with IP buffer. RNA was extracted using TRIzol reagent and quantified by qPCR. For qPCR, 2 µg of RNA was reversely transcribed to cDNA with ReverTra Ace qPCR RT kit (Thermo, USA). Gene expression was quantified using Hieff qPCR SYBR Green Master Mix (Yeasen, China) on CFX Connect Real-Time system (Bio-Rad, USA). Relative expression of target gene was analyzed using established ∆∆Ct threshold method. Primers used in this study was listed in Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA stability assay.\u003c/b\u003e LX-2 cells were treated with Actinomycin D at a final concentration of 4 µg/mL for the indicated time periods and collected. Total RNAs were extracted and analyzed with qPCR. β-actin was used for normalization.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot, immunoprecipitation, immunohistochemistry and immunocytochemistry.\u003c/b\u003e The performance of these experiments were carried out according to previous description\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis.\u003c/b\u003e All data are expressed as mean ± SD. Differences between experimental and control groups were assessed by one-way ANOVA using GraphPad Prism 10. \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cb\u003e1. NAT10 expression is increased in the tissues of hepatic fibrosis and TGF-β1 stimulated LX-2 cells.\u003c/b\u003e Mice were infected with \u003cem\u003eS. japonicum\u003c/em\u003e cercariae to induce liver fibrosis, with the aim to detect NAT10 expression in fibrotic liver tissues. As compared with non-infected mice, NAT10 significantly increased in the livers of \u003cem\u003eS. japonicum\u003c/em\u003e infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Human HSC cell line, LX-2, stimulated by TGF-β1, was used as activated HSC cell model, to detect NAT10 expression. Upon TGF-β1 stimulation, NAT10 increased in LX-2 cells along with the increase of activation markers of HSCs, α-SMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The increased expression of NAT10 and α-SMA in TGF-β1 stimulated LX-2 cells was also detected with Immunofluorescence assay. NAT10 was mainly distributed in the nucleus, especially in the nucleolus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Immunofluorescence assay confirmed that TGF-β1 stimulation enhanced ac4C modification abundance in LX-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). These findings collectively suggested heightened expression of NAT10 in mice fibrotic livers and TGF-β1 activated human LX-2 cells, which provide substantial evidence for a significant correlation of NAT10 mediated ac4C RNA modification with HSC activation and liver fibrosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2. ac4C-seq analysis indicates that TGF-β1 enhances ac4C RNA modification of LX-2 cells and differential acetylated genes are enriched in pathways of hepatic diseases.\u003c/b\u003e ac4C sequencing was performed using mRNA enriched from TGF-β1 stimulated LX-2 cells. Compared with control cells, the mRNA from TGF-β1 activated LX-2 cells showed the enhanced ac4C modification of mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). 11746 acetylated cytosines were detected in TGF-β1 activated LX-2 cells and 8132 sites were detected in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). As reported in human HeLa cells\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, ac4C modification was also enriched in CDS (coding sequence) region of mRNAs from TGF-β1 activated LX-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Motifs centered around ac4C modified cytosines demonstrated the alteration of ac4C located preference sequence in LX-2 cells upon TGF-β1 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). A higher frequency of GGG upstream of the ac4C site was observed in the probability sequence context from TGF-β1 activated LX-2 cells, as compared with control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Differentially acetylated genes were also subjected to KEGG pathway enrichment analysis and significant enrichment for the “Hepatocellular carcinoma”, “Hepatitis B”, “NAFLD” (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) was observed. Collectively, ac4C sequencing analysis indicated the enhanced ac4C mRNA modification in TGF-β1 activated LX-2 cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3. NAT10 inhibition suppresses LX-2 activation upon TGF-β1 stimulation, leading to the transcriptomic alteration.\u003c/b\u003e NAT10 is currently the only protein discovered to catalyze ac4C RNA modification\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, siRNA specific to NAT10 was used to inhibit NAT10 expression, to assess the role of NAT10 to LX-2 activation. The marked elevation of α-SMA was observed in LX-2 cells stimulated with TGF-β1, while NAT10 inhibition resulted in the loss of increase of α-SMA in TGF-β1 stimulated LX-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Enhanced migration capability related with skeletal protein remodeling including vimentin and α-SMA is a typical feature of activated HSCs. The results of Transwell assay demonstrated that the enhanced migration of LX-2 cells upon TGF-β1 stimulation was suppressed by NAT10 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Activated HSCs was also characterized with enhanced proliferation. CCK8 assay indicated that TGF-β1 stimulated LX-2 cells exhibited more increased proliferation, as compared with NAT10 siRNA transfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Transcriptomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) revealed that TGF-β1 stimulation led to the upregulation of 841 genes and the downregulation of 552 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), while in NAT10 siRNA transfected cells, there were 408 genes upregulated and 298 genes downregulated upon TGF-β1 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). In conclusion, the above results suggested that NAT10 inhibition suppressed TGF-β1 induced LX-2 activation, accompanied with transcriptomic alteration.\u003c/p\u003e\u003cp\u003e\u003cb\u003e4. Transcriptomic analysis indicates that NAT10 regulates ECM-receptor interaction in TGF-β1 activated LX-2 cells.\u003c/b\u003e NAT10 was reported to modify various substrates including histones, non-histone proteins, and RNA through acetyl transfer reactions\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Co-immunoprecipitation was performed to detect whether NAT10 acts as protein acetyltransferase and enhancement of acetylation of protein complex combined with NAT10 was not detected upon TGF-β1 stimulation (Supplementary Fig.\u0026nbsp;1), which indicated that NAT10 might primarily function as an RNA acetyltransferase rather than a protein acetyltransferase in LX-2 cells. Differential genes from TGF-β1 stimulated cells (TGFB) and cells transfected with NAT10 siRNA and then stimulated with TGF-β1 (siNAT10_TGFB) were enriched and analyzed. GO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and KEGG analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) indicated that differential genes of TGFB and siNAT10_TGFB are enriched in multiple pathways related with ECM function, including ECM-receptor interaction, which was confirmed by enrichment plot from Gene set enrichment analysis (GSEA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Venny analysis was further performed online using 841 genes upregulated in TGF-β1 activated LX-2 cells (TGFb-DEG-up), 408 genes upregulated in NAT10 siRNA transfected and further TGF-β1 stimulated LX-2 cells (siNAT10-DEG-up) enriched by transcriptomic analysis, 5402 differential genes with enhanced ac4C modification in TGF-β1 activated LX-2 cells enriched by ac4C sequencing analysis (TGFb-ac4C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). 52 genes with enhanced ac4C modification, which was upregulated TGFb-DEG-up, however not upregulated in siNAT10-DEG-up, were regarded as candidate genes regulated by NAT10 via ac4C modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) and input into Metascape online analysis, indicating significant gene enrichment for “Extracellular matix organization” and “ECM proteoglycans” (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Collectively, RNA-seq and ac4C-seq analysis demonstrated that target genes regulated by NAT10 via ac4C RNA modification in TGF-β1 activated LX-2 cells are enriched in ECM-receptor interaction and related pathways .\u003c/p\u003e\u003cp\u003e\u003cb\u003e5. NAT10 regulates TGFB1 and COL1A1 expression via ac4C modification in activated LX-2 cells.\u003c/b\u003e As differential genes from TGFB and siNAT10_TGFB were analyzed, “TGF-beta signaling pathway” was noticed from GSEA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). \u003cem\u003eTGFB1\u003c/em\u003e was enriched in the heatmap of “TGF-beta signaling pathway” (Supplementary Fig.\u0026nbsp;2). In the heatmap of GSEA enrichment plot “ECM-receptor interaction”, \u003cem\u003eCOL1A1\u003c/em\u003e, encoding collagen type I, is the top ranked gene (Supplementary Fig.\u0026nbsp;2). \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e are among the 52 genes selected from the Venny analysis presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, which were regarded as candidate genes regulated by NAT10 via ac4C modification. TGF-β1, an important inflammatory cytokine promoting tissue fibrosis, was used in this study to activate LX-2 cells. We therefore suppose that TGF-β1 enhances NAT10 expression and NAT10 catalyzes ac4C modification of \u003cem\u003eTGFB1\u003c/em\u003e mRNA, to form a positive feedback, promoting ac4C modification and expression of \u003cem\u003eCOL1A1\u003c/em\u003e mRNA. Transcriptional expression of \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e, was detected by qPCR, which indicated that the increased mRNA levels of \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e in TGF-β1 activated LX-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), was suppressed by NAT10 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe Integrated Genomics Viewer (IGV) was used to track and display the distribution of ac4C modified sites detected by ac4C-sequencing. Compared with the control cells, \u003cem\u003eCOL1A1\u003c/em\u003e and \u003cem\u003eTGFB1\u003c/em\u003e mRNA from the TGF-β1 stimulated cells showed more sites with ac4C modification (Supplementary Fig.\u0026nbsp;3). To verify the ac4C sequencing results, ac4C-RIP-qPCR was performed to determine the enhanced ac4C modification of \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e mRNA in TGF-β1 stimulated LX-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). ac4C modification of \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e mRNA was also detected in NAT10 siRNA transfected cells, however, the results indicated “not detectable” due to exceeding the detection range (data not shown).\u003c/p\u003e\u003cp\u003eThe effect of NAT10 to \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e mRNA stability was evaluated by RNA stability assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e). Stability of both \u003cem\u003eCOL1A1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) and \u003cem\u003eTGFB1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) mRNA was enhanced in LX-2 upon TGF-β1 stimulation, while enhanced mRNA stability of these genes was abolished by NAT10 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e). Expression of collagen I and TGFB1 at protein level was further detected and the results were consistent with mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Overall, the above results indicated that in TGF-β1 activated LX-2 cells, NAT10 regulated transcriptional and translational expression of TGFB1 and COL1A1, via ac4C modification.\u003c/p\u003e\u003cp\u003e\u003cb\u003e6. NAT10 expression is increased in activated primary HSCs and NAT10 correlates with\u003c/b\u003e \u003cb\u003eTGFB1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCOL1A1\u003c/b\u003e \u003cb\u003eexpression.\u003c/b\u003e In order to verify the results from human LX-2 cells, primary HSCs were isolated from mice liver and cultured to naturally activate \u003cem\u003ein-vitro\u003c/em\u003e. As primary HSCs were cultivated from 2 to 6 days \u003cem\u003ein-vitro\u003c/em\u003e, the expression of HSC activation marker molecules α-SMA and vimentin increased, indicating cell activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Along with cell activation, the expression of NAT10, TGFB1, and collagen I also increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). NAT10 siRNA was transfected into primary HSCs from mice at 2 days after isolation and seeding, to detect \u003cem\u003eTgfb1\u003c/em\u003e and \u003cem\u003eCol1a1\u003c/em\u003e at mRNA level. In primary HSCs from mice, mRNA expression of \u003cem\u003eNat10\u003c/em\u003e, \u003cem\u003eTgfb1\u003c/em\u003e and \u003cem\u003eCol1a1\u003c/em\u003e in cells cultured to 6 days was enhanced, as compared with cells cultured to 2 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), while in \u003cem\u003eNat10\u003c/em\u003e inhibited cells, mRNA expression of \u003cem\u003eTgfb1\u003c/em\u003e and \u003cem\u003eCol1a1\u003c/em\u003e was inhibited, as compared with non-specific siRNA transfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eNASH (non-alcoholic steatohepatitis) is currently the main cause of liver fibrosis in humans. Activation of HSCs is an important mechanism driving the progression of NASH to liver fibrosis\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. A Gene Expression Omnibus (GEO) dataset\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e related to human NASH, GSE212837, has been selected to evaluate the expression of \u003cem\u003eNAT10\u003c/em\u003e, \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e, and the correlation between these genes. GSE212837 is a single-cell sequencing dataset. According to the marker genes described\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, HSC cell population was separated to analyze the expression of \u003cem\u003eNAT10\u003c/em\u003e, \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e using R language (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.R-project.org/\u003c/span\u003e\u003cspan address=\"http://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). HSCs of NASH group displayed an increased expression of \u003cem\u003eNAT10\u003c/em\u003e, \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e, as compared with control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Linear regression analysis using R showed a strong correlation between \u003cem\u003eNAT10\u003c/em\u003e and \u003cem\u003eTGFB1\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e = 1.000), and a moderate correlation between \u003cem\u003eNAT10\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e (R\u003csup\u003e2\u003c/sup\u003e = 0.425) in the NASH group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). In the Human Liver Proteome Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.liverproteome.org/\u003c/span\u003e\u003cspan address=\"http://www.liverproteome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), TGFB1 and COL1A1 were also retrieved as significantly expressed proteins in liver cirrhosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). To be summarized, the above results based on \u003cem\u003ein-vitro\u003c/em\u003e activated primary HSCs from mice and human NASH GEO dataset analysis indicate that NAT10 expression is increased in activated HSCs and NAT10 correlates with \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e expression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn current study, TGF-β1 activated human LX-2 cells and \u003cem\u003ein-vitro\u003c/em\u003e activated primary hepatic stellate cells from mice were used as activated HSC cell model, to explore the role of NAT10 mediated ac4C RNA modification in the activation of HSCs.\u003c/p\u003e\u003cp\u003eNAT10 catalyzes the acetylation of various substrates including proteins and RNA. Previous studies have determined that NAT10 acts as a lysine acetyltransferase to acetylate α-tubulin, p53 and histones\u003csup\u003e[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. This study did not clarify the increased lysine acetyltransferase activity of NAT10 in LX-2 cells stimulated by TGF-β1. However, the enzyme's activity is highly specific, and this result does not rule out the possibility that NAT10 may act as lysine acetyltransferase to catalyze acetylation of certain specific proteins. Further experiments are essential to clarify the activity of lysine acetyltransferase of NAT10 during HSCs activation.\u003c/p\u003e\u003cp\u003eIn this study, the enhanced expression of NAT10 was determined in liver fibrosis tissue from \u003cem\u003eschistosomiasis japonicum\u003c/em\u003e infected mice and LX-2 human HSCs activated by TGF-β1. In \u003cem\u003ein-vitro\u003c/em\u003e activated primary HSCs from mice, NAT10 was defined to increase at both mRNA level and protein level. Analysis of GSE212837 from a study on human NASH\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e also revealed enhanced expression of NAT10. Recent studies have reported the mechanism by which NAT10 expression is regulated\u003csup\u003e[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. At the transcriptional level, it was reported that transcriptional factor Hif-1 regulates NAT10 expression in gastric cancer cells\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. We have previously reported that Hif\u0026minus;1 promotes HSC activation via regulating its multiple target genes\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Potential transcription factors regulating NAT10 expression were analyzed through the JASPER transcription factor online analysis, which suggested that multiple transcription factors including Hif\u0026minus;1, NRF1, ZNF460 and ZNF284, may regulate NAT10 expression (supplementary Fig.\u0026nbsp;4). In addition to transcriptional regulation, post-translational modification enhances stability of NAT10 at protein level. It was reported that Khib modification (2-hydroxyisobutyrylation) of NAT10 enhances the interaction of NAT10 with deubiquitinase USP39, resulting in increased NAT10 protein stability\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The mechanism by which NAT10 increases in activated hepatic stellate cells deserves further exploration.\u003c/p\u003e\u003cp\u003eTGF-β1/Smad signaling pathway is an important pathway that promotes the HSCs activation\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Analysis of GSE212837 from a study on human NASH\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e revealed a strong correlation between NAT10 and TGFB1 in activated HSCs in NASH. Enhanced expression of NAT10 leads to increased ac4C mRNA modification in LX-2 cells, including enhanced ac4C modification of \u003cem\u003eTGFB1\u003c/em\u003e mRNA, leading to increased RNA stability of \u003cem\u003eTGFB1\u003c/em\u003e, and subsequently sustained enhancement of TGF-β1 protein expression. These results suggested that TGF-β1 and NAT10 form positive feedback through the ac4C modification in activated HSCs. Target genes regulated by NAT10 are enriched in ECM-receptor interaction in TGF-β1 stimulated LX-2 cells. The top gene enriched in \u0026ldquo;ECM-receptor interaction\u0026rdquo; is \u003cem\u003eCOL1A1\u003c/em\u003e, encoding collagen I. Results from LX-2 cells and primary HSCs from mice, verified that \u003cem\u003eCOL1A1\u003c/em\u003e is target gene regulated by NAT10 via ac4C modification.\u003c/p\u003e\u003cp\u003eRecent reports have revealed that NAT10 inhibition suppresses the progression of multiple liver diseases\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In current study, the results revealed that NAT10 acts as ac4C acetyltransferase and forms a positive feedback with TGF-β1 in HSCs, thus modulating the ac4C modification of \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e mRNA, which improves the stability of \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e mRNA, promotes protein expression of TGF-β1 and collagen I, facilitating HSCs activation and the progression of liver fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). Targeting NAT10 regulated TGF-β1-ac4C-\u003cem\u003eCOL1A1\u003c/em\u003e axis might be a promising direction for intervening in liver fibrosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ch4\u003e.ac4C (N4-acetylcytosine); \u0026alpha;-SMA (\u0026alpha;-smooth muscle actin); CDS (coding sequence); CHX (cycloheximide); COL1A1 (collagen type I alpha 1 chain); ECM (extracellular matrix); HCC (hepatocellular carcinoma); HSC (hepatic stellate cell); MASH (metabolic dysfunction-associated steatohepatitis); MASLD (metabolic dysfunction-associated steatotic liver disease); MMPs (matrix metalloproteinases); NASH (non-alcoholic steatohepatitis), NAT10 (N-acetyltransferase 10); NK-\u0026kappa;B (nuclear factor kappa B subunit); PDGF (platelet-derived growth factor); TIMPs (tissue metalloproteinase inhibitors); TGF-\u0026beta;1 (transforming growth factor-beta1); TGFB1 (transforming growth factor-beta1)\u0026nbsp;\u003c/h4\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAZ and CS conceived and designed the project. AZ performed most of the experiments. FG, YZ, JS, NA, XS, QQ, WL performed some of the experiments. AZ, JS, NA and CS analyzed and interpreted the data. AZ and CS wrote the manuscript. All the authors have read this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (No.c, to Shi C), Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, Huazhong University of Science and Technology (No.500153003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank Dr. Lu Wan (Department of Pathophysiology, School of Basic Medicine, Huazhong University of Science and Technology) for kindly reviewing the manuscript. We thank Dr. Yuyu Xie (Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology) and Dr. Zhangbo Cui (School of Public Health, Tongji Medical College, Huazhong University of Science and Technology) for the guidance and assistance of animal study. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis. Lancet. 2014;383(9930):1749-1761. \u003c/li\u003e\n\u003cli\u003eHigashi T, Friedman SL, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev. 2017;121:27-42. \u003c/li\u003e\n\u003cli\u003eHu HH, Chen DQ, Wang YN, et al. New insights into TGF-\u0026beta;/Smad signaling in tissue fibrosis. Chem Biol Interact. 2018;292:76-83. \u003c/li\u003e\n\u003cli\u003eMachnicka MA, Milanowska K, Osman Oglou O, et al. MODOMICS: a database of RNA modification pathways--2013 update. 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Multi-modal analysis of human hepatic stellate cells identifies novel therapeutic targets for metabolic dysfunction-associated steatotic liver disease. J Hepatol. 2025;82(5):882-897. \u003c/li\u003e\n\u003cli\u003eWang S, Li K, Pickholz E, et al. An autocrine signaling circuit in hepatic stellate cells underlies advanced fibrosis in nonalcoholic steatohepatitis. Sci Transl Med. 2023;15(677):eadd3949.\u003c/li\u003e\n\u003cli\u003eJin C, Wang T, Zhang D, et al. Acetyltransferase NAT10 regulates the Wnt/\u0026beta;-catenin signaling pathway to promote colorectal cancer progression via ac4C acetylation of KIF23 mRNA. J Exp Clin Cancer Res. 2022;41(1):345. \u003c/li\u003e\n\u003cli\u003eLarrieu D, Britton S, Demir M, et al. Chemical inhibition of NAT10 corrects defects of laminopathic cells. Science. 2014;344(6183):527-532.\u003c/li\u003e\n\u003cli\u003eLarrieu D, Vir\u0026eacute; E, Robson S, et al. Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway. Sci Signal. 2018;11(537):eaar5401.\u003c/li\u003e\n\u003cli\u003eLiu X, Tan Y, Zhang C, et al. NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2. EMBO Rep. 2016;17(3):349-366.\u003c/li\u003e\n\u003cli\u003eLv J, Liu H, Wang Q, et al. Molecular cloning of a novel human gene encoding histone acetyltransferase-like protein involved in transcriptional activation of hTERT. Biochem Biophys Res Commun. 2003;311(2):506-513.\u003c/li\u003e\n\u003cli\u003eZhang G, Zheng B, Chen X, et al. N4-Acetylcytidine Drives Glycolysis Addiction in Gastric Cancer via NAT10/SEPT9/HIF-1\u0026alpha; Positive Feedback Loop. Adv Sci (Weinh). 2023;10(23):e2300898. \u003c/li\u003e\n\u003cli\u003eLiao L, He Y, Li SJ, et al. Lysine 2-hydroxyisobutyrylation of NAT10 promotes cancer metastasis in an ac4C-dependent manner. Cell Res. 2023;33(5):355-371.\u003c/li\u003e\n\u003cli\u003eLiu J, Xie Y, Cui Z, et al. Bnip3 interacts with vimentin, an intermediate filament protein, and regulates autophagy of hepatic stellate cells. Aging (Albany NY). 2020 Dec 3;13(1):957-972. \u003c/li\u003e\n\u003cli\u003eLiu H, Xu L, Yue S, et al. Targeting N4-acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2-mediated HMGB2 mRNA translation. Cancer Commun (Lond). 2024;44(9):1018-1041.\u003c/li\u003e\n\u003c/ol\u003e\n"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"hepatology-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hepi","sideBox":"Learn more about [Hepatology International](https://www.springer.com/journal/12072)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/hepi/default.aspx","title":"Hepatology International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"hepatic fibrosis, hepatic stellate cells, NAT10, N4-acetylcytosine, TGF-β1, COL1A1","lastPublishedDoi":"10.21203/rs.3.rs-7284137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7284137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eLiver fibrosis is characterized by deposition of excessive extracellular matrix (ECM). The major source of ECM is activated hepatic stellate cells (HSCs). NAT10 is the only known acetyltransferase catalyzing ac4C RNA modification. The purpose of this study is to explore the role of NAT10 acting as ac4C acetyltransferase during HSC activation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eNAT10 was detected in fibrotic liver tissues from \u003cem\u003eS. japonicum\u003c/em\u003e infected mice with immunohistochemistry and TGF-β1 stimulated LX-2 human HSC cells with Western blot, immunofluorescent staining and qPCR. NAT10 was inhibited with specific siRNA in LX-2 cells to detect HSC activation molecular marker with Western blot, cell motility with Transwell assay, cell proliferation with CCK8 assay. ac4C modification was assessed in TGF-β1 stimulated LX-2 cells with immunofluorescent staining. ac4C bisulfite sequencing and transcriptomic sequencing analysis were performed to analyze ac4C modified genes regulated by NAT10 in TGF-β1 stimulated LX-2 cells. Possible target genes regulated by NAT10 were determined using qPCR, ac4C-RIP-qPCR, RNA stability assay, and were further verified using primary hepatic stellate cells from mice and using analysis with GEO datasets.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eNAT10 increases in \u003cem\u003eS. japonicum\u003c/em\u003e infected mice liver and activated HSCs. NAT10 is correlated with \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e expression in activated HSCs and NAT10 inhibition suppresses HSCs activation. NAT10 promotes the ac4C modification and stability of \u003cem\u003eTGFB1\u003c/em\u003e and \u003cem\u003eCOL1A1\u003c/em\u003e mRNA, thus enhancing their protein expression.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eNAT10 acts as ac4C acetyltransferase and forms a positive feedback with TGF-β1 in HSCs, thus modulating the TGF-β1-ac4C-\u003cem\u003eCOL1A1\u003c/em\u003e axis, to promote the HSCs activation and contributes to liver fibrosis.\u003c/p\u003e","manuscriptTitle":"NAT10 promotes the activation of hepatic stellate cells by modulating the TGF-β1-ac4C- COL1A1 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 09:34:42","doi":"10.21203/rs.3.rs-7284137/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-09-12T22:48:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-18T05:44:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-17T07:29:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-06T11:21:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Hepatology International","date":"2025-08-05T11:13:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"hepatology-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hepi","sideBox":"Learn more about [Hepatology International](https://www.springer.com/journal/12072)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/hepi/default.aspx","title":"Hepatology International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"535647a1-7f7f-4a40-9afc-98abc709eb8a","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:00:44+00:00","versionOfRecord":{"articleIdentity":"rs-7284137","link":"https://doi.org/10.1007/s12072-025-10998-x","journal":{"identity":"hepatology-international","isVorOnly":false,"title":"Hepatology International"},"publishedOn":"2026-01-20 15:57:06","publishedOnDateReadable":"January 20th, 2026"},"versionCreatedAt":"2025-08-25 09:34:42","video":"","vorDoi":"10.1007/s12072-025-10998-x","vorDoiUrl":"https://doi.org/10.1007/s12072-025-10998-x","workflowStages":[]},"version":"v1","identity":"rs-7284137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7284137","identity":"rs-7284137","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}