Objective  To identify key genes potentially regulating metabolic dysfunction-associated steatohepatitis (MASH) through comprehensive mining of the Gene Expression Omnibus (GEO) database, validate their functions in vitro and in vivo models, and preliminarily reveal their potential regulatory mechanisms.

Methods  Transcriptional data from mouse [choline deficient diet (CD) group vs. high-fat/high-cholesterol diet (HFHC) group; CD group vs. methionine- and choline deficient diet (MCD) group] and human (healthy vs. MASH) in the GEO database were integrated to screen differentially expressed genes and conduct enrichment analysis. In vitro MASH model [palmitic acid (PA)/oleic acid (OA) co-stimulation of mouse primary hepatocytes] and in vivo model (adeno-associated virus-mediated liver-specific gene knockout Cas9 transgenic mice fed with HFHC for 16 weeks) were constructed. The expression of candidate genes was verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot (WB), and lipid accumulation was evaluated by Nile red staining and lipid detection (triglycerides). Serum indicators including low-density lipoprotein cholesterol (LDL-C), aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH) were detected in vivo, and liver pathological changes were assessed by hematoxylin-eosin staining (HE), picrosirius red (PSR), oil red O (ORO) staining, and immunohistochemistry (IHC). Metabolic pathways were analyzed by RNA sequencing (RNA-seq).

Results  A total of 16 common differentially expressed genes (Ubd, ANAX2, TREM2, LGALS3, CAPG, LPL, CCL2, TOP2A, CD52, EMP3, MKI67, TMSB10, CCDC88A, S100A4, CRIP1, TAGLN2) were screened, among which Ubd had the highest expression level. The mRNA expression level of the Ubd gene was significantly upregulated in clinical MASH patients and mouse MASH models (P<0.05). Overexpression of Ubd in primary mouse hepatocytes aggravates lipid accumulation which is induced by PA/OA stimulation, while knockdown of Ubd can alleviate PA/OA-induced lipid accumulation. In the in vivo MASH model of mice, compared with the control group, the liver tissue of the Ubd knockout group shows significant improvement in lipid accumulation, with a significant reduction in liver ORO staining, a significant decrease in CD11b+ inflammatory cells in liver tissue, and a significant reduction in PSR staining positive areas. Mechanism analysis based on transcriptomics indicated that knockdown of Ubd could lead to significant activation of the fatty acid metabolism pathway, thereby effectively reducing MASH-related lipid accumulation and lipotoxicity.

Conclusion  UBD significantly promotes MASH, possibly by inhibiting fatty acid oxidation and aggravating lipid accumulation, thereby accelerating the progression of MASH disease.

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