Abstract
Background & AimsIn patients with non‐alcoholic fatty liver disease (NAFLD), liver biopsy is the gold standard to detect non‐alcoholic steatohepatitis (NASH) and stage liver fibrosis. We aimed to identify differentially expressed mRNAs and non‐coding RNAs in serum samples of biopsy‐diagnosed mild and severe NAFLD patients with respect to controls and to each other.MethodsWe first performed a whole transcriptome analysis through microarray (n = 12: four Control: CTRL; four mild NAFLD: NAS ≤ 4 F0; four severe NAFLD NAS ≥ 5 F3), followed by validation of selected transcripts through real‐time PCRs in an independent internal cohort of 88 subjects (63 NAFLD, 25 CTRL) and in an external cohort of 50 NAFLD patients. A similar analysis was also performed on liver biopsies and HepG2 cells exposed to oleate:palmitate or only palmitate (cellular model of NAFL/NASH) at intracellular/extracellular levels. Transcript correlation with histological/clinical data was also analysed.ResultsWe identified several differentially expressed coding/non‐coding RNAs in each group of the study cohort. We validated the up‐regulation of UBE2V1, BNIP3L mRNAs, RP11‐128N14.5 lncRNA, TGFB2/TGFB2‐OT1 coding/lncRNA in patients with NAS ≥ 5 (vs NAS ≤ 4) and the up‐regulation of HBA2 mRNA, TGFB2/TGFB2‐OT1 coding/lncRNA in patients with Fibrosis stages = 3‐4 (vs F = 0‐2). In in vitro models: UBE2V1, RP11‐128N14.5 and TGFB2/TGFB2‐OT1 had an increasing expression trend ranging from CTRL to oleate:palmitate or only palmitate‐treated cells both at intracellular and extracellular level, while BNIP3L was up‐regulated only at extracellular level. UBE2V1, RP11‐128N14.5, TGFB2/TGFB2‐OT1 and HBA2 up‐regulation was also observed at histological level. UBE2V1, RP11‐128N14.5, BNIP3L and TGFB2/TGFB2‐OT1 correlated with histological/biochemical data. Combinations of TGFB2/TGFB2‐OT1 + Fibrosis Index based on the four factors (FIB‐4) showed an Area Under the Curve (AUC) of 0.891 (P = 3.00E‐06) or TGFB2/TGFB2‐OT1 + Fibroscan (AUC = 0.892, P = 2.00E‐06) improved the detection of F = 3‐4 with respect to F = 0‐2 fibrosis stages.ConclusionsWe identified specific serum coding/non‐coding RNA profiles in severe and mild NAFLD patients that possibly mirror the molecular mechanisms underlying NAFLD progression towards NASH/fibrosis. TGFB2/TGFB2‐OT1 detection improves FIB‐4/Fibroscan diagnostic performance for advanced fibrosis discrimination.
Highlights
Background & AimsIn patients with non‐alcoholic fatty liver disease (NAFLD), liver biopsy is the gold standard to detect non‐alcoholic steatohepatitis (NASH) and stage liver fibrosis
In order to further investigate the possible link between validated transcripts and cellular/molecular mechanisms associated with dis‐ ease evolution, we evaluated whether RNA deregulation observed in serum of NAFLD patients was present in liver tissue of NAFLD patients and in in vitro models of NAFL and NASH, both at intracel‐ lular and extracellular levels
In this multiphase case‐control study, we performed an initial screening of coding messenger RNA (mRNA) and non‐coding RNA (ncRNA) in biopsy‐proven NAFLD patients at different stages of histopathological severity and con‐ trols (n = 12: 4 CTRL, 4 NAFLD activity score (NAS) ≤ 4 F = 0, 4 NAS ≥ 5 F = 3)
Summary
Non‐alcoholic fatty liver disease (NAFLD) accounts for the most in‐ creasing cause of chronic liver disease, hepatocellular carcinoma and of end‐stage liver disease leading to liver transplantation.[1,2]. Body fluid RNAs, including long non‐coding RNAs (lncRNAs) and messenger RNAs (mRNAs), have many of the essential characteristics of good biomarkers, for ex‐ ample: (i) they are non‐invasive accessible; (ii) their levels can eas‐ ily be determined by basic molecular biology methods, first of all LAY SUMMARY Liver biopsy still remains the gold standard for NAFLD diagnosis confirmation, distinction between simple stea‐ tosis and NASH, and fibrosis staging. | 3 experimental evidence is beginning to characterize the role of spe‐ cific lncRNAs in the pathogenesis of liver fibrosis 20 and several met‐ abolic functions, such as free fatty acid β‐oxidation, lipogenesis and insulin secretion.[21] Numerous studies performed high‐throughput analysis of mRNAs in liver tissues from NAFLD patients or in vivo animal models, which allowed the identification of several pathways associated with disease progression.[22,23,24,25,26] To date, no study has been performed on circulating mRNA and lncRNA signatures in sera from NAFLD patients. The main aim of this work was to analyse the whole transcriptome profile in serum samples of NAFLD biopsy‐ diagnosed patients to identify novel non‐invasive biomarkers which are able to identify NAFLD patients with NASH and/or advanced fibrosis
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