Transcriptomic Profiling Identifies Novel Hepatic and Intestinal Genes Following Chronic Plus Binge Ethanol Feeding in Mice

Abstract

Background: Alcohol-associated liver disease accounts for half of cirrhosis-related deaths worldwide. The spectrum of disease varies from simple steatosis to fibrosis, cirrhosis and ultimately hepatocellular carcinoma. Understanding the disease on a molecular level helps us to develop therapeutic targets.

Aim: We performed transcriptomic analysis in liver and ileum from chronic plus binge ethanol-fed mice, and we assessed the role of selected differentially expressed genes and their association with serum bile acids and gut microbiota.

Methods: Wild-type mice were subjected to a chronic Lieber-DeCarli diet model for 8 weeks followed by one binge of ethanol. RNA-seq analysis was performed on liver and ileum samples. Associations between selected differentially regulated genes and serum bile acid profile or fecal bacterial profiling (16S rDNA sequencing) were investigated.

Results: We provide a comprehensive transcriptomic analysis to identify differentially expressed genes, KEGG pathways, and gene ontology functions in liver and ileum from chronic plus binge ethanol-fed mice. In liver, we identified solute carrier organic anion transporter family, member 1a1 (Slco1a1; encoding for organic anion transporting polypeptides (OATP) 1A1), as the most down-regulated mRNA, and it is negatively correlated with serum cholic acid level. Prokineticin 2 (Prok2) mRNA, a cytokine-like molecule associated with gastrointestinal tract inflammation, was significantly down-regulated in ethanol-fed mice. Prok2 mRNA expression was negatively correlated with abundance of Allobaculum (genus), Coprococcus (genus), Lachnospiraceae (family), Lactococcus (genus), and Cobriobacteriaceae (family), while it positively correlated with Bacteroides (genus).

Conclusions: RNA-seq analysis revealed unique transcriptomic signatures in the liver and intestine following chronic plus binge ethanol feeding.

Keywords: 16S rDNA sequencing; Bile acids; Gene expression; Metabolome; Microbiome.

Figures

Figure 1.. RNA-seq analysis in liver in response to chronic plus binge ethanol: differentially expressed genes (DEGs) and KEGG pathway analysis.

(A) Volcano plot illustrating DEGs in liver between ethanol-fed and control mice (absolute fold change > 2; adjusted P value < 0.05). (B) Number of up- and down-regulated genes in liver in response of ethanol compared with control. (C) Top 20 significant KEGG pathways in ethanol-fed mice compared with controls (q value < 0.05). Control group: n=10; Ethanol group: n=19. For figure 1(B) and 1(C), cutoff for DEGs is absolute fold change > 1.5, adjusted P value < 0.05.

Figure 2.. Gene ontology (GO) biological processes and gene-concept networks of cholesterol-associated GO terms in liver in response to chronic plus binge ethanol.

(A) Bar chart of top 20 significant GO terms of liver differentially expressed genes (DEGs) following ethanol treatment compared with controls. (B) Bar chart of selected and significantly different GO terms following chronic plus binge ethanol feeding. (C) Gene-concept networks of cholesterol metabolic process and cholesterol biosynthetic process. Control group: n=10; Ethanol group: n=19. Significant GO terms cutoff: adjusted P value < 0.05.

Figure 3.. Correlation between hepatic Slco1a1 expression and serum bile acid profiling.

(A) Hepatic mRNA level of Slco1a1 in control and ethanol groups (real-time qPCR). (B) Heatmap of Spearman correlation between hepatic Slco1a1 mRNA and serum bile acids. Red color indicates positive correlation, blue color indicates negative correlation. (C) Spearman correlation between cholic acid and Slco1a1. Control group: n=10; Ethanol group: n=19. *P < 0.05. ****P < 0.0001. Slco1a1, Solute carrier organic anion transporter family, member 1a1; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; MCA, muricholic acid; T-CA, taurocholic acid; T-CDCA, taurochenodeoxycholic acid; T-DCA, taurodeoxycholic acid; T-MCA, tauromuricholic acid.

Figure 4.. RNA-seq analysis in ileum in response to chronic plus binge ethanol: differentially expressed genes (DEGs) and KEGG pathway analysis.

(A) Volcano plot illustrating DEGs in ileum between ethanol-fed and control mice (absolute fold change > 2; adjusted P value < 0.05). (B) Number of up- and down-regulated genes in ileum in response to ethanol compared with control. (C) Top 20 significant KEGG pathways in ethanol-fed mice compared with controls (q value < 0.05). Control group: n=10; Ethanol group: n=19. For figure 1(B) and 1(C), cutoff for DEGs is absolute fold change > 1.5, adjusted P value <0.05.

Figure 5.. Gene ontology (GO) biological processes and gene-concept networks of triglyceride-associated GO terms in ileum in response to chronic plus binge ethanol.

(A) Bar chart of top 20 significant GO terms of ileum differentially expressed genes (DEGs) following ethanol treatment compared with controls. (B) Bar chart of selected and significantly different GO terms following chronic plus binge ethanol feeding. (C) Selected and significantly changed KEGG pathways following chronic plus binge ethanol feeding. (D) Gene-concept networks of triglyceride metabolic process and triglyceride biosynthetic process. Control group: n=10; Ethanol group: n=19. Significant GO terms cutoff: adjusted P value < 0.05.

Figure 6.. Correlation between ileal Prok2 expression and metagenomic (16S rDNA sequencing) data.

(A) mRNA level of ileal Prok2 in control and ethanol groups (real-time qPCR). (B) Heatmap of Spearman correlation between ileal Prok2 mRNA and the 30 most abundant bacteria at genus level, determined from 16S rDNA gene sequencing. Red color indicates positive correlation, blue color indicates negative correlation. (C) Spearman correlation between ileal Prok2 mRNA and Allobaculum abundance. Control group: n=10; Ethanol group: n=19. *P < 0.05, ****P < 0.0001.