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

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.