HNRNPC Promotes Autophagy by Upregulating m6A-Modified FOXO1 Expression and Exacerbates the Progression of Rheumatoid Arthritis

Background Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial hyperplasia and chronic inflammation, whose molecular regulatory mechanisms are not fully elucidated. Both N6-methyladenosine (m6A) RNA modification and cellular autophagy are involved in the pathogenesis and progression of RA, but their synergistic regulatory mechanisms remain unclear. Objective To determine the expression profile of HNRNPC in RA and verify whether HNRNPC upregulates FOXO1 expression by stabilizing m6A-modified FOXO1 mRNA, thereby activating autophagy in MH7A cells and promoting RA inflammatory progression. Methods Bioinformatics analysis was performed based on the RA-related dataset GSE93776 from the GEO database to screen differentially expressed m6A regulators and construct a diagnostic model. An in vitro RA cell model was established by stimulating human synovial fibroblast cell line MH7A with tumor necrosis factor-α (TNF-α). MeRIP-qPCR was used to detect the m6A modification level of FOXO1 mRNA. RIP-qPCR was performed to validate the direct binding between HNRNPC and FOXO1 mRNA. siRNA-mediated knockdown of HNRNPC or overexpression of FOXO1 was employed, combined with qRT-PCR, Western blot, CCK-8, and immunofluorescence techniques to analyze the regulatory effects of HNRNPC on FOXO1 expression, MH7A cell viability, and autophagy. Rescue experiments were conducted to verify the specificity of this regulatory axis. Results ① Bioinformatics analysis revealed that HNRNPC was significantly overexpressed in RA and was the most crucial m6A regulator for distinguishing RA from normal samples (highest Mean Decrease Gini value). ② In the RA cell model, both mRNA and protein expression levels of HNRNPC and FOXO1 were significantly upregulated. The m6A modification level of FOXO1 mRNA was increased compared to the control group. Knockdown of HNRNPC significantly reduced the m6A modification level of FOXO1. ③ RIP experiments confirmed that HNRNPC could directly bind to FOXO1 mRNA. Actinomycin D experiments indicated that HNRNPC knockdown shortened the half-life of FOXO1 mRNA, reducing its stability and protein expression. ④ Knockdown of HNRNPC inhibited autophagy activation in MH7A cells, manifested as decreased LC3-II/I ratio and Beclin1 expression, reduced number of autophagic flux fluorescent puncta, and suppression of abnormal MH7A cell proliferation. Rescue experiments confirmed that overexpression of FOXO1 could partially reverse the autophagy inhibition and decreased cell viability caused by HNRNPC knockdown. Conclusion This study reveals for the first time a novel regulatory axis in RA: HNRNPC ↑ → m6A-modified FOXO1 mRNA stabilization → FOXO1 expression ↑ → MH7A cell autophagy activation → RA inflammatory progression ↑. It identifies HNRNPC as a key m6A regulator in RA, providing a new molecular target and theoretical basis for targeted RA therapy.