Screening and validation of Hub genes in bile acid-induced hepatocyte response based on transcriptome sequencing

Abstract

Objective To screen and verify the Hub genes involved in conjugated bile acid-induced hepatocyte metabolism through transcriptome sequencing analysis. Methods Primary mouse hepatocytes were treated with taurocholic acid (TCA), and the gene expression of TCA-induced hepatocytes was detected by RNA-Seq analysis. Differential expression genes (DEGs) were identified using a fold change ≥1.5 and P value < 0.05 as the screening criteria. The protein-protein interaction (PPI) network of DEGs was constructed using the STRING database, and Hub genes were screened using Cytoscape software based on the PPI network. Real-time qPCR was used to validate the identified Hub gene in vitro and in vivo. Western blotting was employed to detect the expression of proteins related to PSAT1/AKT/GSK3β signaling pathway. The glycogen level in the hepatocytes and liver tissues were detected using glycogen detection kit. Results RNA-Seq analysis identified 293 DEGs in primary mouse hepatocytes upon TCA treatment, including 122 up-regulated genes and 171 down-regulated genes. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Gene Set Enrichment Analysis (GSEA) revealed that these DEGs were mainly enriched in metabolism, inflammatory and tumorigenic pathways. Cytohubba plugin identified 16 Hub genes, including Ccnd1, Ppara, Irs1, Ccl2, Ddit3, Serpine1, Nr0b2, Atf3, Phgdh, Trib3, Lepr, G6pc, Psat1, Cxcl2, Asns, and Fgf21. RT-qPCR results demonstrated that various conjugated bile acids, glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), and TCA can induce a significant increase in Psat1 mRNA level in primary mouse hepatocytes (P < 0.05). In vivo experiments also showed that the mRNA level of Psat1 was increased significantly in the liver tissues of mouse cholestatic model induced by bile duct ligation (BDL) when compared to the control group (P < 0.05). Western blotting indicated that TCA treatment induced the expression of Psat1 and activation of AKT/GSK3β signaling pathway in primary mouse hepatocytes. TCA treatment also resulted in an increase in glycogen synthesis in primary mouse hepatocytes, which was consistent with an increase in glycogen synthesis in the liver tissue and a decrease in blood glucose level in the mouse model established with BDL (P < 0.05). Conclusion Psat1 may be a gene involved in alteration of glucose metabolism induced by bile acid, and its high expression may be associated with these changes in glucose metabolism in hepatocytes.

Keywords

• cholestasis
• glucose metabolism
• primary mouse hepatocytes
• rna-sequencing