Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease worldwide, but a reliable non-invasive method to quantify liver steatosis in primary healthcare is not available. Circulating microRNAs have been proposed as biomarkers of severe/advanced NAFLD (steatohepatitis and fibrosis). However, the use of circulating miRNAs to quantitatively assess the % of liver fat in suspected NAFLD patients has not been investigated. We performed global miRNA sequencing in two sets of samples: human livers from organ donors (n = 20), and human sera from biopsy-proven NAFLD patients (n = 23), both with a wide range of steatosis quantified in their liver biopsies. Partial least squares (PLS) regression combined with recursive feature elimination (RFE) was used to select miRNAs associated with steatosis. Moreover, regression models with only 2 or 3 miRNAs, with high biological relevance, were built. Comprehensive microRNA sequencing of liver and serum samples resulted in two sets of abundantly expressed miRNAs (418 in liver and 351 in serum). Pearson correlation analyses indicated that 18% of miRNAs in liver and 14.5% in serum were significantly associated with the amount of liver fat. PLS-RFE models demonstrated that 50 was the number of miRNAs providing the lowest error in both liver and serum models predicting steatosis. Comparison of the two miRNA subsets showed 19 coincident miRNAs that were ranked according to biological significance (guide/passenger strand, relative abundance in liver and serum, number of predicted lipid metabolism target genes, correlation significance, etc.). Among them, miR-10a-5p, miR-98-5p, miR-19a-3p, miR-30e-5p, miR-32-5p and miR-145-5p showed the highest biological relevance. PLS regression models with serum levels of 2−3 of these miRNAs predicted the % of liver fat with errors <5%.
Keywords: NAFLD patient screening; NAFLD patient stratification; circulating miRNAs; non-alcoholic fatty liver disease; non-invasive; steatosis quantification.