Alcohol Use Disorder Causes Profound Global Changes in Gene Expression and Splicing in the Liver

Objective Alcohol use disorder (AUD) is a widespread illness commonly leading to alcoholic liver disease (ALD) and cirrhosis with an increased incidence of hepatocellular carcinoma (HCC), but the mechanisms of alcohol-related oncogenesis in the liver are incompletely understood. Splicing represents a mechanism of messenger RNA processing, and its impairment is implicated in various cancers. We tested the hypothesis that AUD predisposes to ALD and HCC via dysregulation of splicing. Methods RNA sequencing was performed on liver biopsies from patients with different stages of alcoholic liver disease: early alcoholic steatohepatitis (eASH), non-severe alcoholic hepatitis (nsAH), and severe alcoholic hepatitis (sAH); furthermore, explants were collected from patients who underwent liver transplantation due to sAH (exAH). Alignment was performed with the STAR aligner (v.2.5.2a) against the hg19 human genome. Raw counts were processed by edgeR in Bioconductor and normalized using CPM values. Resulting bam files from the STAR alignment were indexed with samtools for use by rMATS for the analysis of missplicing events. For exon expression, DEXSeq R package was employed, with GENCODE v.19 serving as a reference. Expression of small nuclear RNAs (snRNAs) and splicing factors was measured by real-time PCR. Data on oncogenes and tumor suppressors implicated in HCC as well as RNA sequencing files from liver of patients with HCC and HCC + AUD were retrieved from The Cancer Genome Atlas. Computational analysis of the affected pathways was performed using Gene Ontology Consortium, Gene Set Enrichment Analysis, Oncogene Database, and LncRNA Ontology databases. Results We found that alcohol caused widespread changes in transcriptome in all stages of ALD: among ~ 58,000 analyzed genomic features, ~ 4,900 were altered in eASH, ~ 9,100 – in nsAH, 14,100 – in sAH, and ~ 14,300 – in exAH. We observed thousands of missplicing events in all hepatic conditions, with mutually exclusive exons (MEE) being the most common event and exon skipping (ES) – second most common event. Analysis of ~ 600,000 exons revealed that ALD is associated with a genome-wide effect on exon expression, with ~ 50,000 exons being differentially expressed in eASH, ~ 130,000 – in nsAH, ~ 150,000 – in sAH, and ~ 120,000 – in exAH. To determine whether alcohol directly perturbs splicing, we subjected rats to alcohol vapor for 7 weeks and measured hepatic expression of two components of major and minor spliceosomes: snRNAs and splicing factors. Expression of such snRNAs as snU2, snU6atac, and snU11 was drastically decreased, while expression of splicing factors was not affected. Analysis of aberrantly expressed hepatic long non-coding RNAs (lncRNAs) from patients with AUD and patients with HCC + AUD demonstrated that overlapping lncRNAs were functionally related to splicing and spliceosome. Screening of 26 oncogenes and tumor suppressors, commonly involved in HCC pathogenesis, revealed that AUD affected the hepatic expression and/or splicing of most of these cancer-related genes. Conclusions Alcohol causes profound genome-wide changes in gene expression and splicing in the liver, likely via affecting the spliceosome and splicing-related lncRNAs. This results in altered expression and missplicing of key oncogenes and tumor suppressors involved in HCC, suggesting a novel mechanism of oncogenesis in the liver of patients with AUD.