Integrated Machine Learning and SHAP Analysis Identifies TRIM21 Ubiquitination of HSPA8 Driving P

ABSTRACT

Metabolic dysfunction–associated fatty liver disease (MAFLD) is a metabolic disorder characterized by excessive hepatic lipid accumulation. Despite its rapidly increasing global prevalence, the molecular mechanisms driving MAFLD initiation and progression remain incompletely understood. Identification of key regulatory molecules may provide novel diagnostic and therapeutic targets. Key pathogenic genes associated with MAFLD were identified using machine‐learning approaches integrated with SHapley Additive exPlanations (SHAP) analysis. Spearman correlation analysis was performed to assess the relationships between HSPA8 expression and immune cell infiltration, inflammatory cytokine levels, and lipid metabolism–related gene expression. An in vitro MAFLD model was established by treating HepG2 cells with oleic acid (OA) and palmitic acid (PA). Western blotting and co‐immunoprecipitation assays were conducted to validate the interaction between TRIM21 and HSPA8. A noninvasive diagnostic model for MAFLD was constructed using a random forest (RF) algorithm based on seven endoplasmic reticulum–related genes ( HSPA8, CDKN1A, CEBPB, BBC3, EGR1, FOS, and SCD ). SHAP‐based interpretability analysis demonstrated that these genes collectively contributed to model performance, with HSPA8 exerting the strongest influence. HSPA8 expression was significantly upregulated in MAFLD and showed strong correlations with NAFLD activity score, immune cell infiltration, inflammatory cytokine secretion, and lipid metabolism–related gene expression. Suppression of HSPA8 markedly attenuated lipid accumulation in OA‐ and PA–treated HepG2 cells. Mechanistically, TRIM21 was identified as an upstream E3 ubiquitin ligase that promoted K48‐linked polyubiquitination of HSPA8, thereby facilitating its ubiquitin–proteasome–mediated degradation. HSPA8 was identified as a central pathogenic driver in MAFLD, while dysregulation of the E3 ubiquitin ligase TRIM21 exacerbated disease progression by destabilizing HSPA8. These findings highlight the TRIM21–HSPA8 axis as a potential diagnostic and therapeutic target in MAFLD.