Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: Diagnostic and mechanistic relevance

Abstract

Background & aims: Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages.

Methods: We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR.

Results: Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages, but miR-193a-5p consistently showed increased levels in all comparisons. Relative to NAFL/non-alcoholic steatohepatitis (NASH) with mild fibrosis (stage 0/1), 3 miRNAs (miR-193a-5p, miR-378d, and miR378d) were increased in cases with NASH and clinically significant fibrosis (stages 2-4), 7 (miR193a-5p, miR-378d, miR-378e, miR-320b, miR-320c, miR-320d, and miR-320e) increased in cases with NAFLD activity score (NAS) 5-8 compared with lower NAS, and 3 (miR-193a-5p, miR-378d, and miR-378e) increased but 1 (miR-19b-3p) decreased in steatosis, activity, and fibrosis (SAF) activity score 2-4 compared with lower SAF activity. The significant findings for miR-193a-5p were replicated in the additional cohort with NAFLD. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n = 80); liver GPX8 levels correlated positively with serum miR-193a-5p.

Conclusions: Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD.

Lay summary: MicroRNAs (miRNAs) are small pieces of nucleic acid that may turn expression of genes on or off. These molecules can be detected in the blood circulation, and their levels in blood may change in liver disease including non-alcoholic fatty liver disease (NAFLD). To see if we could detect specific miRNA associated with advanced stages of NAFLD, we carried out miRNA sequencing in a group of 183 patients with NAFLD of varying severity together with 10 population controls. We found that a number of miRNAs showed changes, mainly increases, in serum levels but that 1 particular miRNA miR-193a-5p consistently increased. We confirmed this increase in a second group of cases with NAFLD. Measuring this miRNA in a blood sample may be a useful way to determine whether a patient has advanced NAFLD without an invasive liver biopsy.

Keywords: ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUROC, area under the receiver operating characteristic; Biomarker; CPM, counts per million; Ct, cycle threshold; ER, endoplasmic reticulum; FC, fold change; FIB-4, fibrosis-4; FLIP, fatty liver inhibition of progression; GTEx, Genotype-Tissue Expression; MicroRNA; NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; NASH, non-alcoholic steatohepatitis; Non-alcoholic fatty liver disease; PCA, principal component analysis; SAF, steatosis–activity–fibrosis; Sequencing; TGF-β, transforming growth factor-beta; cDNA, complementary DNA; logFC, log2 fold change; miRNA, microRNA; qPCR, quantitative PCR.

Graphical abstract

Fig. 1

Principal component analysis plot for the sequencing data. Quality controlled, filtered and batch corrected miRNA sequencing data within the plot were coloured according to histological group to aid the identification of clustering by either the first or second principal component. miRNA, microRNA; NASH, non-alcoholic steatohepatitis; PC1, principal component 1; PC2, principal component 2.

Fig. 2

Replication by qPCR of miR-193a-5p and miR-3687 associations. Levels of miR-193a-5p are shown for (A) significant fibrosis (NASH F2–F4) relative to minimal fibrosis (NAFL–NASH F0/F1) (n = 359), (B) advanced NAS (NAS 5–8) relative to mild NAS (NAS 1–4) (n = 359), and (C) advanced SAF activity (SAF activity 3-4) relative to mild SAF activity (SAF activity 0-2) (n = 359). Levels of miR-3687 are shown for (D) significant fibrosis (NASH F2–F4) relative to minimal fibrosis (NAFL–NASH F0/F1) (n = 371), (E) advanced NAS (NAS 5–8) relative to mild NAS (NAS 1–4) (n = 371), and (F) advanced SAF activity (SAF activity 3–4) relative to mild SAF activity (SAF activity 0–2) (n = 371). 2^-ΔΔCt was calculated relative to controls for all samples. Median values are shown with 95% CIs. Mann–Whitney U tests were performed for all comparisons. NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; NASH, non-alcoholic steatohepatitis; qPCR, quantitative PCR; SAF, steatosis–activity–fibrosis.

Fig. 3

Differentially expressed predicted target genes of miR-193a-5p in liver RNA-seq. (A) RNA-seq data from liver tissue were analysed for 3 comparisons: advanced NAS (NAS 5–8) relative to mild NAS (NAS 1–4), advanced SAF activity (SAF activity 3–4) relative to mild SAF activity (SAF activity 0–2), and significant fibrosis (NASH F2–F4) relative to minimal fibrosis (NAFL–NASH F0/F1). The genes that overlapped with those predicted by in silico tools to be targets of miR-193a-5p are shown in the heatmap. Hierarchical clustering is based on levels of fold change in gene expression in the liver tissue. Statistical significance in the RNA-seq data was determined using a Benjamini–Hochberg adjusted p value ≤0.05: ∗p ≤0.05, ∗∗p ≤0.01, and ∗∗∗p ≤0.001. (B and C) Linear models of 80 overlapping NAFLD samples between the miRNA-seq and RNA-seq datasets for miR-193a-5p and (B) GPX8 and (C) COL1A1. AFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; NASH, non-alcoholic steatohepatitis; SAF, steatosis–activity–fibrosis.

Fig. 4

In vitro functional assessment of miR-193a-5p expression in Hep G2 cells. Hep G2 cells were treated with fatty acids (oleic acid [500 μM], palmitic acid [250 μM], or a combination of oleic [500 μM] and palmitic acid [250 μM]) for 24 h. All treatments were performed in triplicate, and qPCRs of each were performed in triplicate. Data were normalised to the control condition (untreated) using the 2^-ΔΔCt method. An unpaired Student’s t-test was performed for all conditions relative to the control conditions (∗∗p ≤0.01). Data are presented as the mean with error bars representing the SEM. qPCR, quantitative PCR.