Novel hepatic microRNAs upregulated in human nonalcoholic fatty liver disease.

Jarkko Soronen,Pirkka-Pekka Laurila,Alexander Sigruener,Julia Perttilä,Sanja Sädevirta,Marja Leivonen,Hannele Yki-Järvinen,Antti-Pekka Sarin,You Zhou,Vesa M Olkkonen,Ksenia Sevastianova,Gerd Schmitz

doi:10.14814/phy2.12661

Abstract

MicroRNAs (miRNAs) control gene expression by reducing mRNA stability and translation. We aimed to identify alterations in human liver miRNA expression/function in nonalcoholic fatty liver disease (NAFLD). Subjects with the highest (median liver fat 30%, n=15) and lowest (0%, n=15) liver fat content were selected from >100 obese patients for miRNA profiling of liver biopsies on microarrays carrying probes for 1438 human miRNAs (a cross-sectional study). Target mRNAs and pathways were predicted for the miRNAs most significantly upregulated in NAFLD, their cell-type-specific expression was investigated by quantitative PCR (qPCR), and the transcriptome of immortalized human hepatocytes (IHH) transfected with the miRNA with the highest number of predicted targets, miR-576-5p, was studied. The screen revealed 42 miRNAs up- and two downregulated in the NAFLD as compared to non-NAFLD liver. The miRNAs differing most significantly between the groups, miR-103a-2*, miR-106b, miR-576-5p, miRPlus-I137*, miR-892a, miR-1282, miR-3663-5p, and miR-3924, were all upregulated in NAFLD liver. Target pathways predicted for these miRNAs included ones involved in cancer, metabolic regulation, insulin signaling, and inflammation. Consistent transcriptome changes were observed in IHH transfected with miR-576-5p, and western analysis revealed a marked reduction of the RAC1 protein belonging to several miR-576-5p target pathways. To conclude, we identified 44 miRNAs differentially expressed in NAFLD versus non-NAFLD liver, 42 of these being novel in the context of NAFLD. The study demonstrates that by applying a novel study set-up and a broad-coverage array platform one can reveal a wealth of previously undiscovered miRNA dysregulation in metabolic disease.

Highlights

Nonalcoholic fatty liver disease (NAFLD) is a disorder characterized by hepatic fat accumulation due to nonalcoholic causes (Perry et al 2014)
Blood samples were taken for measurement of complete blood cell count and concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, triglycerides, glucose, C-peptide, and insulin, as previously described (Kotronen et al 2007)
We focused on the eight miRNAs differing most significantly between the two study groups (P < 0.05 after Bonferroni correction)

Summary

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a disorder characterized by hepatic fat accumulation due to nonalcoholic causes (Perry et al 2014) This burgeoning health problem is closely associated with all features of the metabolic syndrome (Yki-J€arvinen 2014), and its prevalence increases rapidly worldwide with the expanding epidemic of obesity (Bellentani et al 2010). MiR-34a, which regulates the sirtuin 1/p53 pathway inhibiting the synthesis and stimulating the b-oxidation of fatty acids, was shown to be upregulated in subjects with NAFLD, its activity correlating with disease severity (Castro et al 2013). Peroxisome proliferator-activated receptor a (PPARa), a transcription factor with a pivotal role in hepatic lipid metabolism, oxidative stress, inflammatory response, and fibrogenesis, was shown to be targeted in hepatoma cells by miR-21, and correlated negatively with this miRNA species in human liver (Kida et al 2011). Peroxisome proliferator-activated receptor a (PPARa), a transcription factor with a pivotal role in hepatic lipid metabolism, oxidative stress, inflammatory response, and fibrogenesis, was shown to be targeted in hepatoma cells by miR-21, and correlated negatively with this miRNA species in human liver (Kida et al 2011). miR-296-5p, which modulates lipoapoptosis, was demonstrated to be reduced in the liver of subjects with advanced NAFLD as compared to patients with simple steatosis or healthy controls (Cazanave et al 2011)

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