m6A Modification Destabilizes Prss8 and Activates Hepatic Stellate Cells via TLR4-Mediated Inflammatory Responses.

Abstract

The 6-methyladenine (m6A) modification plays a major role in various diseases. Serine protease 8 (Prss8) contributes to the initiation and progression of liver fibrosis (LF). However, the mechanism by which the m6A modification of Prss8 induces hepatic stellate cells (HSCs) activation in the LF is unclear. This study focused on exploring the contribution of Prss8 m6A modification to the pathogenesis of LF. First, primary hepatic parenchymal cells (hepatocytes) and HSCs were isolated from a mouse model of LF, and a coculture of these two types of cells was used as the object of study. Then, real-time fluorescence quantitative PCR, methylated RNA immunoprecipitation, and Western blotting were used to test the expression levels of Prss8 mRNA and protein, Prss8 m6A modification, Collagen I, α-SMA, and TLR4. Finally, the expression levels of inflammatory markers were measured via an enzyme-linked immunosorbent assay. Compared with the control group, the model group presented significantly lower Prss8 mRNA and protein levels in hepatocytes but greater levels of Prss8 m6A modification; moreover, the expression of HSC activation markers and the TLR4, IL-1β, and IL-18 proteins was significantly elevated. Mutation of the Prss8 m6A modification site led to upregulation of Prss8 mRNA and protein and decreased levels of m6A modification, TLR4, IL-1β, and IL-18. Furthermore, mutation of the Prss8 m6A modification site increased the stability of Prss8 mRNA. Rescue experiments confirmed the regulatory link between Prss8 m6A modification and TLR4. Overall, Prss8 m6A modification decreases the stability of its mRNA, promoting TLR4-mediated inflammatory cascades and leading to excessive activation of HSCs. Targeting Prss8 m6A modification is a promising therapeutic strategy for LF.