MicroRNAs Signature Panel Identifies Heavy Drinkers with Alcohol-Associated Cirrhosis from Heavy Drinkers without Liver Injury

SIMPLE SUMMARY: Alcohol-associated liver disease (ALD) is a preventable disease if identified early, but current biomarkers lack specificity to identify who amongst drinkers will progress to advanced disease, i.e., cirrhosis. MicroRNAs are small molecules that play a regulatory role in several diseases, are affected by alcohol and may be important players in alcohol use disorders, such as ALD. So far, microRNAs associated with ALD are based on comparisons with non-drinking healthy controls. Our new approach was to compare global miRNA profiles in heavy drinking patients without liver injury versus heavy drinkers with cirrhosis to identify miRNAs specific to liver disease. Our novel discoveries include (i) the identification of a set of microRNAs that differentiated drinkers with cirrhosis from those without liver injury; (ii) an inverse relationship of microRNAs expression with disease severity, suggesting their potential use in monitoring disease progression; (iii) the identification of microRNA target pathways involved in cell death, inflammation, fibrosis and fat metabolism; (iv) and the association of some microRNAs with known genetic risks involved in triglyceride metabolism, underscoring the growing role of lipids (fats) in ALD. This study advances knowledge in the field by identifying new microRNAs as potential diagnostic and/or therapeutic targets. ABSTRACT: Background: Alcohol-associated liver disease (ALD) is the most common disorder of prolonged drinking. Mechanisms underlying cirrhosis in such patients remain unclear. MicroRNAs play regulatory role in several diseases, are affected by alcohol and may be important players in alcohol use disorders, such as cirrhosis. Methods: We investigated serum samples from heavy chronic alcohol users (80 g/day (male) and 50 g/day (female) for ≥10 years) that were available from our previously reported GenomALC study. A subset of GenomALC drinkers with liver cirrhosis (cases, n = 24) and those without significant liver disease (drinking controls, n = 23) were included. Global microRNA profiling was performed using high-throughput real-time quantitative PCR to identify the microRNA signatures associated with cirrhosis. Ingenuity Pathway Analysis (IPA) software was utilized to identify target mRNAs of significantly altered microRNAs, and molecular pathways were analysed. Identified microRNAs were analysed for correlation with traditional liver disease biomarkers and risk gene variants previously reported from GenomALC genome-wide association study. Results: The expression of 21 microRNAs was significantly downregulated in cases compared to drinking controls (p < 0.05, ∆∆Ct > 1.5-fold). Seven microRNAs (miR-16, miR-19a, miR-27a, miR-29b, miR-101, miR-130a, and miR-191) had a highly significant correlation (p < 0.001) with INR, bilirubin and MELD score. Three microRNAs (miR-27a, miR-130a and miR-191) significantly predicted cases with AUC-ROC 0.8, 0.78 and 0.85, respectively (p < 0.020); however, INR performed best (0.97, p < 0.001). A different set of six microRNAs (miR-19a, miR-26a, miR-101, miR-151-3p, miR-221, and miR-301) showed positive correlation (ranging from 0.32 to 0.51, p < 0.05) with rs10433937:HSD17B13 gene variant, associated with the risk of cirrhosis. IPA analysis revealed mRNA targets of the significantly altered microRNAs associated with cell death/necrosis, fibrosis and increased steatosis, particularly triglyceride metabolism. Conclusions: MicroRNA signatures in drinkers distinguished those with liver cirrhosis from drinkers without liver disease. We identified mRNA targets in liver functions that were enriched for disease pathogenesis pathways.