The NSUN2-ALYREF/YBX1-PTK7 axis drives hepatic stellate cell activation in liver fibrosis through m5C mRNA modification

Liver fibrosis results from the overproduction and deposition of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). NSUN2 is the main methyltransferase responsible for mRNA m 5 C modification. ALYREF and YBX1 are binding proteins recognizing m 5 C-modified mRNA. This study delineates the functional relevance of the NSUN2-m 5 C-ALYREF/YBX1 axis in HSC activation. NSUN2, ALYREF and YBX1, as well as m 5 C modification were detected in liver tissues from multiple fibrotic mouse models, in TGF-β1 stimulated LX-2 human HSCs and primary HSCs isolated from mice with immunohistochemistry, Western blot and immunofluorescent staining. The expression of NSUN2, ALYREF and YBX1 was inhibited using specific shRNAs or overexpressed in LX-2 cells to assess HSC activation markers by Western blot and cell motility by Transwell assay. m 5 C modification was evaluated using dot blot. m 5 C-modified genes regulated by NSUN2 were identified with m 5 C bisulfite sequencing and transcriptomic analysis. Potential genes regulated by NSUN2, ALYREF and YBX1 were identified with qPCR, RIP-qPCR, RNA stability assays, and functional validation. NSUN2, ALYREF and YBX1, as well as m 5 C modification increase in the liver tissues from high-fat diet (HFD) mice, carbon tetrachloride (CCl 4 ) -treated mice, S. japonicum- infected mice and in activated HSCs. Inhibition of NSUN2, ALYREF and YBX1 suppresses HSC activation. NSUN2 promotes m 5 C modification and stabilizes PTK7 mRNA, thereby enhancing PTK7 protein expression and HSC activation. The regulation of PTK7 expression by NSUN2 depends on ALYREF and YBX1. NSUN2-ALYREF/YBX1-PTK7 axis drives HSC activation through m 5 C mRNA modification, which reveals a previously unrecognized epigenetic mechanism governing HSC biology.