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

Katherine Johnson; Peter J Leary; Olivier Govaere; Matthew J Barter; Sarah H Charlton; Simon J Cockell; Dina Tiniakos; Michalina Zatorska; Pierre Bedossa; M Julia Brosnan; Jeremy F Cobbold; Mattias Ekstedt; Guruprasad P Aithal; Karine Clément; Jörn M Schattenberg; Jerome Boursier; Vlad Ratziu; Elisabetta Bugianesi; Quentin M Anstee; Ann K Daly; LITMUS Consortium Investigators§; LITMUS Consortium Investigators

doi:10.1016/j.jhepr.2021.100409

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

JHEP Rep. 2021 Nov 25;4(2):100409. doi: 10.1016/j.jhepr.2021.100409. eCollection 2022 Feb.

Authors

Katherine Johnson¹, Peter J Leary², Olivier Govaere¹, Matthew J Barter³, Sarah H Charlton³, Simon J Cockell^{2

3}, Dina Tiniakos¹, Michalina Zatorska¹, Pierre Bedossa¹, M Julia Brosnan⁴, Jeremy F Cobbold⁵, Mattias Ekstedt⁶, Guruprasad P Aithal⁷, Karine Clément^{8

9}, Jörn M Schattenberg¹⁰, Jerome Boursier¹¹, Vlad Ratziu^{8

9}, Elisabetta Bugianesi¹², Quentin M Anstee^{1

13}, Ann K Daly¹; LITMUS Consortium Investigators§; LITMUS Consortium Investigators

Collaborators

James Clark, Heather J Cordell, Rebecca Darlay, Christopher P Day, Tim Hardy, Yang-Lin Liu, Fiona Oakley, Jeremy Palmer, Rachel Queen, Kristy Wonders, Patrick M Bossuyt, Adriaan G Holleboom, Hadi Zafarmand, Yasaman Vali, Jenny Lee, Karine Clement, Raluca Pais, Detlef Schuppan, Michael Allison, Sergio Rodriguez Cuenca, Vanessa Pellegrinelli, Michele Vacca, Antonio Vidal-Puig, Tuulia Hyötyläinen, Aidan McGlinchey, Matej Orešič, Partho Sen, Jose Mato, Óscar Millet, Jean-Francois Dufour, Stephen Harrison, Stefan Neubauer, Michael Pavlides, Ferenc Mozes, Salma Akhtar, Rajarshi Banerjee, Matt Kelly, Elizabeth Shumbayawonda, Andrea Dennis, Charlotte Erpicum, Manuel Romero-Gomez, Rocío Gallego-Durán, Isabel Fernández, Morten Karsdal, Diana Leeming, Mette Juul Fisker, Elisabeth Erhardtsen, Daniel Rasmussen, Per Qvist, Antonia Sinisi, Estelle Sandt, Maria Manuela Tonini, Maurizio Parola, Chiara Rosso, Fabio Marra, Amalia Gastaldelli, Sven Francque, Stergios Kechagias, Hannele Yki-Järvinen, Kimmo Porthan, Saskia van Mil, George Papatheodoridis, Helena Cortez-Pinto, Luca Valenti, Salvatore Petta, Luca Miele, Andreas Geier, Christian Trautwein, Paul Hockings, Phil Newsome, David Wenn, Cecília Maria Pereira Rodrigues, Rémy Hanf, Pierre Chaumat, Christian Rosenquist, Aldo Trylesinski, Pablo Ortiz, Kevin Duffin, Carla Yunis, Melissa Miller, Theresa Tuthill, Judith Ertle, Ramy Younes, Leigh Alexander, Rachel Ostroff, Mette Skalshøi Kjær, Lars Friis Mikkelsen, Clifford Brass, Lori Jennings, Maria-Magdalena Balp, Miljen Martic, Guido Hanauer, Sudha Shankar, Richard Torstenson, Céline Fournier, Richard Ehman, Michael Kalutkiewicz, Kay Pepin, Joel Myers, Diane Shevell, Gideon Ho, Henrik Landgren, Rob Myers, Lynda Doward, Diane Whalley, James Twiss

Affiliations

¹ Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
² Bioinformatics Support Unit, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
³ Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.
⁴ Internal Medicine Research Unit, Pfizer Inc., Cambridge, MA, USA.
⁵ Oxford Liver Unit, NIHR Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, UK.
⁶ Division of Diagnostics and Specialist Medicine, Department of Health, Medicine, and Caring Sciences, Linköping University, Linköping, Sweden.
⁷ NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and University of Nottingham, Nottingham, UK.
⁸ Institute of Cardiometabolism and Nutrition, Pitié Salpêtrière Hospital, Paris, France.
⁹ Assistance Publique - Hopitaux de Paris, Paris, France.
¹⁰ Metabolic Liver Research Program, I. Department of Medicine, University Medical Center of Johannes Gutenberg-University Mainz, Mainz, Germany.
¹¹ Hepatology Department, Angers University Hospital, Angers, France.
¹² Division of Gastroenterology, Department of Medical Sciences, University of Turin, Turin, Italy.
¹³ Newcastle NIHR Biomedical Research Centre, Newcastle upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, UK.

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.

Abstract

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