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

Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: Diagnostic and mechanistic relevance Authors 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, on behalf of the LITMUS Consortium Investigators Correspondence [email protected] (A.K. Daly), [email protected] (Q.M. Anstee). Graphical abstract Advanced fibrosis Increased miR-193a-5p miR-378d, e High SAF activity score Increased miR-193a-5p miR-378d, e Decreased miR-19b-3p Serum miRNA profiled by RNA sequencing Serum miRNA in discovery NAFLD cohort Replication of miR-193a-5p signal Relevance of serum miR-193a-5p to hepatic GPX8 gene expression Increased miR-193a-5p miR-378d, e miR-320b, c, d, e High NAS score 0 2 4 6 p = 0.0004 NAFL-NASH F1 n = 166 NASH F2-F4 n = 193 2-∆∆Ct 0 2 4 6 p = 0.0012 NAS 1-4 n = 194 NAS 5-8 n = 165 2-∆∆Ct 0 4 6 2 p = 0.0055 SAFact 0-2 n = 215 SAFact 3-4 n = 144 2-∆∆Ct Slope = 0.35 p value = 0.021 -2 -1 0 1 2 3 8910 miR-193a-5p miRNA Voom Batch-corrected counts GPX8 mRNA voom counts Highlights Serum miRNA was sequenced in 183 NAFLD cases of varying severity and 10 population controls. Plasma levels of miR-193a-5p were significantly increased in patients with advanced fibrosis, high NAS scores, or high SAF scores. Other miRNAs including miR378d and miR378e were also significantly increased in certain comparisons. The findings for miR-193a-5p were replicated in a cohort of 372 additional NAFLD cases. 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. https://doi.org/10.1016/j.jhepr.2021.100409 Research article Increased serum miR-193a-5p during non-alcoholic fatty liver disease progression: Diagnostic and mechanistic relevance Katherine Johnson, 1 Peter J. Leary, 2 Olivier Govaere, 1 Matthew J. Barter, 3 Sarah H. Charlton, 3 Simon J. Cockell, 2,3 Dina Tiniakos, 1 Michalina Zatorska, 1 Pierre Bedossa, 1 M. Julia Brosnan, 4,† Jeremy F. Cobbold, 5 Mattias Ekstedt, 6 Guruprasad P. Aithal, 7 Karine Clément, 8,9 Jörn M. Schattenberg, 10 Jerome Boursier, 11 Vlad Ratziu, 8,9 Elisabetta Bugianesi, 12 Quentin M. Anstee, 1,13, * ,‡ Ann K. Daly 1, * ,‡ , on behalf of the LITMUS Consortium Investigators § 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 JHEP Reports 2022. https://doi.org/10.1016/j.jhepr.2021.100409 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: SerummiR-193a-5plevelscorrelatestronglywithNAFLDactivitygradeandfibrosisstage.MiR-193a-5pmayhavea role in the hepatic response to oxidative stress and is a potentialclinically tractable circulating biomarkerfor 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 levelsin blood maychange in liverdisease including non-alcoholic fatty liverdisease (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. © 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: MicroRNA; Non-alcoholic fatty liver disease; Biomarker; Sequencing. Received 10 June 2021; received in revised form 8 October 2021; accepted 9 November 2021; available online 25 November 2021 † Deceased. ‡ These authors contributed equally. *Corresponding authors. Address: Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Fourth Floor, William Leech Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK, Tel.: +44-191-208-7031 (A.K. Daly); Tel.: +44-191-208-7012 (Q.M. Anstee). E-mail addresses: a.k[email protected] (A.K. Daly), [email protected] (Q.M. Anstee). Research article Introduction Non-alcoholic fatty liver disease (NAFLD) affects approximately one-quarter of the adult general population and is the most common cause of liver disease in the Western world. 1 The complex, multisystem disease is characterised by intrahepatic triglyceride accumulation in the absence of excess alcohol intake. 1,2 In a significant minority of patients, NAFLD can progress from steatosis (non-alcoholic fatty liver [NAFL]) to its inflammatory form (non-alcoholic steatohepatitis [NASH]) with hepatocyte injury and progressive fibrosis and may lead to cirrhosis, hepatocellular carcinoma, and endstage liver disease. 3 NAFLD pathogenesis is multifactorial, and so, in addition to metabolic comorbidities and environmental influences, genetic and epigenetic factors confer an increased risk of NAFLD-associated end-stage liver disease. 4,5 Inflammatory disease severity may be assessed histologically using 1 of 2 widely adopted semiquantitative scoring systems: the NASH Clinical Research Network NAFLD activity score (NAS), which conflates the degree of steatosis with steatohepatitic activity grade, and the fatty liver inhibition of progression (FLIP) steatosis–activity–fibrosis (SAF) score, which preserves the distinction between steatosis and activity. 6,7 Histological scoring of a liver biopsy remains the current reference standard for grading and staging NAFLD for clinical trials, even though sampling error as well as inter- and intra-observer discrepancies are well recognised to increase variability and result in misclassification. 8,9 Consequently, circulating biomarkers have been sought to circumvent the need for invasive biopsies. MicroRNAs (miRNAs) are small (22 nucleotides in length) non-coding RNA molecules that can post-transcriptionally regulate gene expression. 10 Mature miRNAs, complexed with facilitative proteins (i.e. the Argonaute protein family), exert their effects by binding mRNA molecules and inducing mRNA degradation or inhibiting translation. 11 Currently, over 2,000 known human miRNAs are recorded on the miRBase database, 12 some of which have been characterised as diagnostic biomarkers for diseases such as cardiovascular disease 13 and cancer. 14 Despite several studies postulating the association of various miRNAs with NAFLD, very few have been unequivocally replicated and validated. 15 The lack of consensus may in part be caused by differences in study design, relatively small numbers of cases being studied, technical approaches, and variances within the chosen techniques. The most abundantly expressed miRNA known in adult liver, miR-122-5p, is possibly the strongest candidate NAFLD biomarker, having been consistently corroborated in independent studies. 16–18 More recently, levels of another miRNA, miR-34a-5p, have been shown to increase in serum as NAFLD progresses, and miR-34a-5p has been incorporated in an in vitro diagnostic test to assist in the identification of patients with fibrosing steatohepatitis. 19 Our aim was to identify additional serum miRNAs biologically relevant to NAFLD pathophysiology and progression with the capacity to serve as biomarkers. We report a comprehensive and unbiased profile of over 2,000 miRNAs in a large international cohort of patients with histologically characterised NAFLD, with separate discovery and replication performed using diverse techniques in independent patient cohorts. Materials and methods Patient recruitment and sample collection The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Cases were derived from the European NAFLD Registry (NCT04442334) with the collection and use of NAFLD patient data and samples approved by the relevant ethical committees in the participating centres and all patients having provided informed consent. 20 The collection of blood samples from ‘healthy’controls (no reported comorbidities or evidence of NAFLD) was approved by the County Durham and Tees Valley Research Ethics Committee (12/NE/012), and informed consent was given by all donors. Disease stage and grade of NAFLD samples were histologically proven by liver biopsy, with the serum samples taken within 6 months of said biopsy. Liver biopsy specimens were scored centrally by 2 expert liver pathologists (DT and PB) to ensure consistency in the analysis and interpretation of histology data. NAFLD was defined by the presence of steatosis in >5% of hepatocytes. Cases were divided into those with NAFL, if steatosis only or steatosis with lobular inflammation was present, and those with NASH, which is characterised by the presence of both lobular inflammation and hepatocellular ballooning in addition to steatosis. 21,22 NAS was calculated as the unweighted sum of steatosis, ballooning, and lobular inflammation, whereas SAF activity was calculated as the unweighted sum of hepatocyte ballooning and lobular inflammation. 6,7 NAFLD activity was graded as low (NAS 1–4or SAF activity 0–2) and high (NAS 5–8 or SAF activity 3–4), and fibrosis staged as none/minimal fibrosis stage (F0–F1) and clinically significant (F2–F4). Serum sample RNA extraction, library preparation, sequencing, and quality control Serum samples (15 l l) were processed through the HTG EdgeSeq platform (HTG Molecular Diagnostics, Inc., Tucson, AZ, USA) using the EdgeSeq miRNA Whole Transcriptome Assay according to the manufacturer’s instructions. Briefly, the samples were lysed with a 1:1 ratio of HTG biofluids lysis buffer to extract RNA, and the lysates input into nuclease protection assays run on the HTG EdgeSeq processor. Sequence adapters were added to the samples in a 16-cycle PCR. PCR products were purified using AMPure XP beads (Beckman Coulter, High Wycombe, UK) and quantified using a KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA). The barcoded libraries were pooled and 2,083 miRNAs (plus 5 negative controls, 1 positive control and 13 housekeeping genes) sequenced at the Genomics Core Facility, Newcastle University, UK, using an Illumina NextSeq 500 System (Illumina Inc., Cambridge, UK) according to the manufacturer’s instructions (single end run, 50 bp). FASTQ files were processed by the pre-installed HTG EdgeSeq parser software to align the probe sequences to the results, giving an output of raw count data. Quality control was conducted using the raw counts according to HTG guidelines. Delta mean values within 2 standard deviations (calculated by the manufacturer as 4.8 for serum samples) were considered acceptable. miRNA sequencing data analysis Data for the 2,083 sequenced miRNAs were analysed using RStudio version 1.0.143 (RStudio: Integrated Development for R. RStudio, Inc., Boston, MA, USA, http://www.rstudio.com/). Raw Research article 2JHEP Reports 2022 vol. 4 j100409 counts were first transformed into counts per million (CPM), and the miRNAs with a CPM < −100 were filtered out of the raw count dataset. DESeq2 version 1.18.1 (https://bioconductor.org/ packages/release/bioc/html/DESeq2.html) 23 was used to generate a dataset –corrected for batch, age, and centre –from which principal component analysis (PCA) plots were constructed. Outliers were defined by visualisation of the PCA plots and removed from the dataset. Comparisons between each of the histological groupings (control, NAFL, NASH-F0/F1, NASH-F2, NASH-F3, and NASH-F4) and various subgroups were performed using limma version 3.34.9 (https://bioconductor.org/packages/ release/bioc/html/limma.html). 24 Statistical significance was determined using a Benjamini–Hochberg-adjusted pvalue < −0.05; no log 2 fold change (logFC) threshold was set. Area under the receiver operating characteristic (AUROC) curves were generated using IBM SPSS Statistics for Windows, version 24 (IBM Corp., Armonk, NY, USA), and significance between 2 AUROC curves was assigned based on the DeLong, DeLong, and Clarke-Pearson method 25 in RStudio version 1.0.143 (RStudio: Integrated Development for R. RStudio, Inc., Boston, MA, USA, http://www. rstudio.com/). miRNA analysis in the replication cohort Total RNA extraction for analysis by quantitative reverse transcriptase PCR Total RNA was extracted from serum using the MACHEREYNAGEL NucleoSpin ® miRNA Plasma Kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany; distributed by Fisher Scientific UK, Loughborough, UK) according to the manufacturer’s instructions with only slight modifications to the protocol. Serum (130 l l) was vortexed with 90 l l Buffer MLP and incubated for 3 min at room temperature. As processing controls,100 fmol of a 50 phosphorylated exogenous synthetic cel-miR-39-3p and 100 fmol of a non-phosphorylated exogenous synthetic cel-miR-39- 3p were spiked-in (50-UCACCGGGUGUAAAUCAGCUUG-30; Integrated DNA Technologies, Inc., Coralville, IA, USA). Total RNA was eluted in 30 l l nuclease-free water and stored at -80C. Complementary DNA synthesis and quantitative reverse transcriptase PCR Using the TaqMan TM Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, Paisley, UK), complementary DNA (cDNA) was synthesised from 3 l l total RNA by reverse transcriptase according to the manufacturer’s instructions. The miR- Amp reaction product was diluted 1:10 in nuclease-free water and stored at -20C. The expressions of 3 miRNAs, including the phosphorylated spike-in, were quantified using pre-formulated TaqMan TM Advanced miRNA Assays (Thermo Fisher Scientific, Table 1. Clinical data for the NAFLD serum samples used in sequencing and quantitative PCR replication. Sequencing cohort Replication cohort pvalue(n = 183) (n = 372) Patient demographic and clinical characteristics Sex (% female) 40% 41% 0.79 Age in years (median [IQR]) 54 (44–60) 56 (47–64) 0.007 BMI (median [IQR]) 31.6 (27.7–36.1) 31.5 (28.4–36.5) 0.46 AST in IU/L (median [IQR]) 37 (27–52) 42 (30–60) 0.014 ALT in IU/L (median [IQR]) 53 (36–79) 57 (38–84) ns Histological characteristics Fibrosis grade <0.0001 F0 59 (32%) 43 (12%) F1 21 (11%) 127 (34%) F2 47 (26%) 66 (18%) F3 38 (21%) 92 (25%) F4 18 (10%) 44 (12%) NASH Yes 128 (70%) 315 (85%) <0.0001 No 55 (30%) 57 (15%) NAS score >4 0.93 Yes 83 (45%) 171 (46%) No 100 (55%) 201 (54%) SAF activity score >2 0.52 Yes 67 (37%) 147 (40%) No 116 (63%) 225 (60%) Values of pfor continuous or categorical datasets were estimated by the Mann–Whitney test and Chi-square/Fisher’s test, respectively. ALT, alanine aminotransferase; AST, aspartate aminotransferase; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; NASH, non-alcoholic steatohepatitis; ns, not significant; SAF, steatosis–activity–fibrosis. -20 -10 0 10 20 -20 20 PC1: 35.55% variance PC2: 14.53% variance Histological group Control NASH F0F1 NASH F2 NASH F3 NASH F4 Simple steatosis Steatosis 0 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, micro- RNA; NASH, non-alcoholic steatohepatitis; PC1, principal component 1; PC2, principal component 2. 3JHEP Reports 2022 vol. 4 j100409 Paisley, UK): 478293_mir (for the spiked-in cel-miR-39-3p), 477954_mir (hsa-miR-193a-5p), and 477855_mir (hsa-miR-122- 5p). Each quantitative PCR (qPCR) mixture contained 5 l l diluted cDNA, 1× TaqMan TM Fast Advanced Master Mix (Thermo Fisher Scientific, Paisley, UK), and 1× TaqMan TM Advanced miRNA Assay, and was made up to 15 l l with nuclease-free water. TaqMan TM MicroRNA Reverse Transcription Kits (Thermo Fisher Scientific, Paisley, UK) were used to synthesise cDNA from 5 l l total RNA according to the manufacturer’s instructions for miRNAs where analysis with material generated via the Advanced miRNA Kit was unsuccessful owing to low levels. The reverse transcription reaction products were diluted 1:10 in nuclease-free water and stored at -20C. The expressions of 3 miRNAs, including the non-phosphorylated spike-in, were quantified using pre-formulated standard TaqMan TM MicroRNA Assays (Thermo Fisher Scientific, Paisley, UK): 000200 (cel-miR- 39-3p), 464645_mat (hsa-miR-3687) and 000426 (hsa-miR-34a- 5p). Each qPCR reaction mixture contained 2.5 l l diluted cDNA, 1 × TaqMan TM Fast Advanced Master Mix (Thermo Fisher Scientific, Paisley, UK), and 1× TaqMan TM Small RNA Assay (Thermo Fisher Scientific, Paisley, UK) and was made up to 20 l l with nucleasefree water. Table 3. miRNAs showing significantly different levels in NASH grouped by fibrosis stage. F2 F3 F4 miRNA logFC Adj. pval. miRNA logFC Adj. pval. miRNA logFC Adj. pval. miR-193a-5p 0.41 4.77E-02 miR-193a-5p 0.72 2.00E-04 miR-193a-5p 0.88 2.32E-03 miR-19b-3p -0.79 4.77E-02 miR-378d 0.48 4.97E-03 miR-4484 0.63 1.44E-02 miR-3135a -0.46 4.77E-02 miR-378e 0.43 1.32E-02 miR-378g 0.51 1.79E-02 miR-378d 0.35 4.77E-02 miR-4484 0.45 1.32E-02 miR-6873-3p -1.24 3.08E-02 miR-378e 0.35 4.77E-02 miR-378a-3p 0.38 1.50E-02 miR-4534 -0.46 4.77E-02 miR-6780a-5p 0.82 2.59E-02 miR-649 -0.59 4.77E-02 miR-670-3p 0.37 4.77E-02 miR-6769a-5p 0.22 4.77E-02 miR-6852-5p 0.27 4.77E-02 miR-25-3p -0.61 4.83E-02 miR-330-3p 0.25 4.83E-02 miR-548ay-5p -0.51 4.83E-02 miR-548d-5p -0.63 4.83E-02 miRNA levels were assessed by sequencing and are shown relative to NAFL. Adjusted pvalue threshold < −0.05. F0/F1 showed no significant differences to NAFL. Adj. pval., adjusted pvalue; logFC, log 2 fold change; miRNA, microRNA; NAFL, non-alcoholic fatty liver; NASH, non-alcoholic steatohepatitis. Table 2. Differences in miRNA levels detected in the NAFLD-only sequencing analysis. miRNA logFC logFC SE Adj. pval.* NASH grouped relative to NAFL miR-193a-5p 0.49 0.11 3.70E-03 miR-378d 0.37 0.09 4.97E-03 miR-378e 0.35 0.09 1.01E-02 miR-4484 0.35 0.09 2.05E-02 NASH with significant fibrosis (F2–F4) relative to NAFL/NASH with minimal fibrosis (F0/F1) miR-193a-5p 0.43 0.10 1.79E-03 miR-378e 0.34 0.07 1.79E-03 miR-378d 0.33 0.07 1.85E-03 High NAS (NAS 5–8) relative to low NAS (NAS 1–4) miR-193a-5p 0.44 0.09 2.61E-04 miR-378d 0.29 0.07 7.14E-03 miR-320d 0.32 0.09 2.39E-02 miR-378e 0.26 0.07 2.39E-02 miR-320b 0.32 0.09 2.89E-02 miR-320c 0.32 0.09 2.89E-02 miR-320e 0.29 0.08 2.89E-02 High SAF activity (SAF activity 3–4) relative to low SAF activity (SAF activity 0–2) miR-193a-5p 0.43 0.11 2.70E-02 miR-378d 0.34 0.09 2.70E-02 miR-378e 0.34 0.09 2.70E-02 miR-19b-3p -0.74 0.20 2.73E-02 Adj. pval., adjusted pvalue; logFC, log2 fold change; miRNA, microRNA; NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; NASH, non-alcoholic steatohepatitis; SAF, steatosis–activity–fibrosis. * Adjusted pvalue significance threshold < −0.05. Research article 4JHEP Reports 2022 vol. 4 j100409 qPCR procedure and analysis of replication cohort data All qPCRs were run in triplicate in MicroAmp TM Fast Optical 96- Well Reaction Plates (Thermo Fisher Scientific, Paisley, UK) on a QuantStudio TM 5 Real-Time PCR machine (Thermo Fisher Scientific, Paisley, UK). PCR cycling conditions were 50C for 2 min and 95C for 20 s, followed by 40 cycles of 95C for 1 s and 60C for 20 s. A reference control, made of pooled cDNA, for each assay was run in triplicate on every plate, meaning the raw cycle threshold (C t ) values could be corrected for batch effects. The batch corrected data were analysed per miRNA by the 2 -DDCt method. 26 Data were plotted using GraphPad Prism version 8.3.1 (GraphPad Software, San Diego, CA, USA; www.graphpad.com). Statistical analyses were performed using the Mann–Whitney U and Kruskal–Wallis 1-way ANOVA tests. Target gene predictions and bioinformatic searches Two online databases were searched for predicted gene targets of miR-193a-5p, namely, TargetScan 27 and miRDB, 28 and the overlapping genes between the 2 were identified. Common genes were identified between the resulting predicted genes and the differentially expressed genes in NAFLD liver as described by Govaere et al. 29 Normalised and transformed counts of miR- 193a-5p, GPX8, and COL1A1 were correlated for the 80 overlapping samples for which miRNA-seq and RNA-seq data were available; a linear model was performed to obtain the slopes and pvalues of the correlations. Single-cell RNA-seq data for GPX8 and COL1A1 were extracted from the Liver Cell Atlas gene browser on 13 August 2020. 30 Data on gene expression were obtained from the Gene Page of the Genotype-Tissue Expression (GTEx) Portal (GTEx Analysis Release V8; dbGaP Accession phs000424.v8.p2). Hep G2 cell culture and treatment The human hepatoblastoma Hep G2 cell line (ATCC ® HB- 8065 TM , Middlesex, UK), was cultured as described previously. 29 To assess effects on miRNA expression, cells were treated for 24 h with the following: (i) 1% bovine serum albumin-conjugated palmitic acid (250 l M), (ii) 1% bovine serum albumin-conjugated oleic acid (500 l M), (iii) 1% bovine serum albumin-conjugated palmitic and oleic acid (250/500 l M) combined solution, and (iv) 1% bovine serum albumin as a lipid-loading control. All treatments were performed in 0 2 4 6 0.5 0.6 0.7 0 4 6 0.5 0.6 0.7 2 A D p = 0.0004 p = 0.0055 NAFL-NASH F1 n = 166 NASH F2-F4 n = 193 NAFL-NASH F1 n = 170 NASH F2-F4 n = 201 0 2 4 6 0.5 0.6 0.7 p = 0.0012 NAS 1-4 n = 194 NAS 5-8 n = 165 NAS 1-4 n = 201 NAS 5-8 n = 170 SAFact 0-2 n = 215 SAFact 0-2 n = 225 SAFact 3-4 n = 146 2-∆∆Ct SAFact 3-4 n = 144 B C E F 2-∆∆Ct 2-∆∆Ct 2-∆∆Ct 2-∆∆Ct 2-∆∆Ct 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 -DDCt 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, nonalcoholic steatohepatitis; qPCR, quantitative PCR; SAF, steatosis–activity–fibrosis. 5JHEP Reports 2022 vol. 4 j100409 triplicate. Total RNA was isolated from the cells as described previously, 29 and cDNA was synthesised and qPCR performed as described above using the TaqMan TM Advanced miRNA cDNA Synthesis Kit and TaqMan TM Advanced miRNA Assay for miR- 193a-5p. Results Study cohort description The current study comprised cases that were histologically characterised and represented the full spectrum of NAFLD severity from NAFL to NASH F4 (cirrhosis). The samples were divided between discovery (n = 183) and replication (n = 372) cohorts. Discovery cases to cover the range of NAFLD phenotypes were selected from those already recruited within the European NAFLD Registry 20 by July 2018 with replication cases selected from the larger cohort available by June 2019. Demographic and clinical data are reported in Table 1. There were some significant differences between the cohorts. In particular, the discovery cohort included a relatively large number of NAFL cases without detectable fibrosis or inflammation to allow detailed comparisons between early and late disease. The replication cohort was significantly older with more severe disease and included a wider spectrum of phenotypes. To obtain baseline measurements for comparison with cases with NAFLD, 10 individuals from a healthy control group (3 male, 7 female, mean age 31 years) were included in the analysis with the discovery cohort. A further 15 control individuals (9 male, 6 female, mean age 42 years) were included in the replication cohort analyses. Comparison of NAFLD serum miRNA profiles with controls The serum miRNA profiles of 183 patients across the NAFLD spectrum and 10 population controls were generated using the HTG EdgeSeq and Illumina NextSeq technologies. Complete sequence data are available as GSE185062 on Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). Initial PCA confirmed that there was no clustering by centre, plate/batch, or age, indicating that batch correction was effective (Fig. S1). PCA showed that the control samples appeared to cluster together, suggesting they had a different miRNA profile to the NAFLD samples, although some overlap was observed (Fig. 1). Relative to controls, levels of 275 serum miRNAs were different in NAFLD patients, 165 increasing and 110 decreasing (Table S1). The most significantly different miRNA was miR- CB A Slope = 0.35 p value = 0.021 -2 -1 0 1 2 3 8910 miR-193a-5p miRNA Voom Batch-corrected counts GPX8 mRNA voom counts Slope = 0.29 p value = 0.074 3 4 5 6 7 8910 miR-193a-5p miRNA Voom Batch-corrected counts Histological group NASH F0F1 NASH F2 NASH F3 NASH F4 Simple steatosis Steatosis COL1A1 PCDHA12 SLC7A1 NETO2 GPX8 ZNF827 GOLGA6A SLC4A10 KCNH1 MTRNR2L8 LDB3 PCDHA10 GABBR2 OLA1 CRYBG3 PCDHA3 RASSF2 PCDHA5 IFFO2 PCDHA4 FADS1 DSCAML1 PCDHA7 XK RAP2A GOLGA6B MTRNR2L1 TNFRSF19 PCDHA11 PCDHAC1 MTRNR2L10 PCDHAC2 MTUS2 GNAQ SLC30A5 TMTC3 NLN C18orf25 UBE2D2 MMP16 CEP57 SRSF10 ZBTB18 TRAF3 PCDHA1 PCDHA6 DNTTIP2 GBA2 PIP4K2B PCDHA9 IPPK SPRED3 MEIS1 BEAN1 PCDHA13 BMF GOLGA6D CUL3 ACVR1 ZMYM4 CUL4A KIAA0100 ANGEL1 GANAB POLDIP2 ELP5 LTB4R WIZ NUP210 IGF2 ANKS1A ITSN1 KMT2A PCDH10 NAS SAF activity Fibrosis * * ** * ** * ** ** * *** ** ** -0.5 0 0.5 1 ** * *** **** *** *** * * * *** COL1A1 mRNA voom counts 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 pvalue < −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. Research article 6JHEP Reports 2022 vol. 4 j100409 3687 for which decreased levels in NAFLD were seen (fold change [FC] 0.10, p=4.57×10 -28 )(Table S2)Whencaseswith NASH and different stages of fibrosis were compared with the population controls, in excess of 200 significant differences in miRNA levels were detected for each fibrosis stage, with the top 200 for each listed in Table S2. The most significant alteration was again the decrease seen for miR-3687 but for NASH F0/F1, F2, and F3 only. Comparison of NAFLD miRNA profiles with NAFL as baseline We next conducted an analysis amongst NAFLD cases only, excluding population controls. Using NAFL as the baseline, we initially looked for serum miRNA changes in NASH cases generally; 4 miRNAs showed increased levels with no miRNAs decreased (Table 2). The miRNA showing the largest increase was miR-193a-5p (FC 1.34, p= 3.70×10 -03 ). We subsequently characterised the miRNA profiles of NASH cases with particular stages of fibrosis (F0–F4) against NAFL (Table 3). The miRNA profile for NASH-F0/F1 was similar to that for NAFL; thereafter, progressive changes in miRNA levels were seen for cases with NASH-F2, NASH-F3, and NASH-F4 (cirrhosis), suggesting that it was the stage of fibrosis rather than grade of steatohepatitic activity that was driving the association. All changes seen in F3 and F4 were increases in miRNA levels, but for F2, both increases and decreases in levels were detected. MiR-193a-5p showed the most significant change with a progressive increase in levels from F2 (FC 1.32) via F3 (FC 1.65) to F4 (FC 1.84) (Table 3). These increases in miR-193a-5p levels with increasing fibrosis score were also in line with those seen for the NAFLD cases compared with population controls (Table S2). To assess the potential for distinguishing between mild and more clinically significant disease, we undertook subgroup comparisons of the miRNA profile in relation to disease activity and fibrosis stage. Three miRNAs showed increased levels in clinically significant fibrosis compared with minimal disease (Table 2); the most statistically significant of these was miR- 193a-5p (FC 1.35, p= 1.79×10 -03 ). When the grade of steatohepatitis was considered using NAS and SAF activity, 7 and 4 miRNAs respectively showed altered levels, with all but 1 of these changes an increase with increasing disease severity (Table 2). MiR-193a-5p again showed the most significant increase (NAS FC = 1.36, p= 2.61×10 -04 ; SAF activity FC = 1.35, p= 2.70×10 -02 ). In light of its association with disease severity, we evaluated miR-193a-5p as a potential circulating biomarker of disease stage and activity by assessing its diagnostic performance in the sequencing dataset using AUROC statistics. AUROCs were also calculated and plotted for miR-122-5p as well as simple composite scores (the aspartate aminotransferase/alanine aminotransferase [AST/ALT] ratio and the fibrosis-4 [FIB-4] score). 31 Relevant curves and values are detailed in Fig. S2. The AUROC for miR-193a-5p was 0.92 for discriminating NAFLD from population controls, whereas the values for differentiating mild disease from significant fibrosis (F0–1vs.F2–4), high NAS, and high SAF activity were 0.68, 0.73, and 0.71, respectively. MiR- 193a-5p significantly outperformed AST/ALT ratio when discriminating between mild vs. significant fibrosis (p= 0.04), high NAS (p= 6.00×10 -05 ), and high SAF activity (p= 0.03). It also had better discriminatory ability than FIB-4 as an indicator of high NAS (p= 3.40×10 -03 ) and exhibited comparable performance with FIB4 for fibrosis. The discriminatory ability of miR- 193a-5p was better than that of miR-122-5p for fibrosis (p= 0.019), NAS (p= 0.012), and SAF activity (p= 1.53×10 -03 ). Replication of miR193a-5 and previously reported miRNAs as predictors of NAFLD progression using qPCR in an independent cohort of samples We sought to replicate selected findings from our sequencing studies relating to miR-193-5p and miR-3687 and additionally quantify serum levels of 2 miRNAs (miR-122-5p and miR-34a- 5p) that have previously been reported as relevant to NAFLD, 16–19 using the independent replication cohort of 372 NAFLD cases; healthy control samples (n = 15) were also included. Limited volumes of serum and inability to analyse using the Advanced miRNA Kit prevented replication of other interesting miRNA signals. Consistent with the results of our sequencing analysis, miR-193a-5p showed significantly higher levels in NAFLD cases with greater disease severity (i.e. more significant fibrosis and high NAS/SAF activity) (Fig. 2). However, the differences observed in miR-3687 levels did not vary significantly with grade or stage of disease in the replication cohort (Fig. 2). Levels of miR-34a-5p were also significantly increased with a higher grade of NAFLD activity (by both NAS and SAF activity) and with more significant fibrosis (Fig. S3). In contrast, miR-122-5p levels did not show significance by fibrosis stage, but levels increased significantly in higher grades of NAFLD activity by both NAS and SAF activity (Fig. S3). Correlation of miR-193a-5p target gene predictions with differential hepatic gene expression measured by RNA-seq in NAFLD In view of the significant increase in serum miR-193a-5p levels seen as disease activity increased and its strong discriminatory ability, we sought to investigate the functional significance of miR-193a-5p in disease progression. Bioinformatic analysis using TargetScan 27 and miRDB 28 predicted that 204 and 327 genes, respectively, were targets of miR-193a-5p, with an overlap of 78 genes between both prediction sets. To better examine the transcriptional correlates of miR-193a-5p, we reanalysed our Untreated Oleic acid Palmitic acid Oleic acid + palmitic acid 0.0 0.5 1.0 1.5 2.0 Fold change relative to untreated cells ** 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 l M], palmitic acid [250 l M], or a combination of oleic [500 l M] and palmitic acid [250 l M]) 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 -DDCt method. An unpaired Student’st-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. 7JHEP Reports 2022 vol. 4 j100409 recently reported hepatic transcriptomics dataset from 206 histologically characterised NAFLD cases to identify genes differentially expressed between mild and severe steatohepatitis and/ or fibrosis. 29 We then projected the list of 78 a priori predicted miR-193a-5p targets onto these data. The resulting differentially expressed genes for each comparison are detailed in Fig. 3. Nine predicted gene targets of miR-193a-5p were significantly differentially expressed in the liver of patients with greater steatohepatitic activity by NAS (COL1A1,CRYBG3,GPX8,OLA1,RAP2A, SLC7A1,XK,ANKS1A, and IGF2) and 3 by SAF activity (COL1A1, GPX8, and IFFO2), and 11 gene targets were differentially expressed in the liver of patients with significant fibrosis (COL1A1,GPX8,IFFO2,NETO2,PCDHA12,SLC7A1,ZNF827,GOLGA6A,ITSN1,KCNH1, and KMT2A). Additionally, the correlation between serum miR-193a-5p and levels in the liver of the 2 genes that were differentially expressed in all 3 comparisons, COL1A1 and GPX8, was investigated in a subgroup of 80 samples for which both serum miRNA-seq and liver RNA-seq data were available (Fig. 3). A significant positive correlation between miR- 193a-5p and GPX8 was observed (slope = 0.35, p= 0.021), and there was a similar trend with COL1A1 (slope = 0.29, p= 0.074). Moreover, single-cell RNA-seq data extracted from the Liver Cell Atlas gene browser 30 showed that both GPX8 and COL1A1 followed a similar pattern of increased expression in cirrhotic compared with uninjured tissue in the mesenchyme (Fig. S4). Quantification of miR-193a-5p expression in a hepatoma cell line We utilised data from the GTEx Portal to visualise the tissuespecific expression of miR-193a-5p, confirming that –in addition to the renal medulla, omentum and subcutaneous adipose tissue, mammary tissue, and cultured fibroblasts –miR-193a-5p was expressed in the liver (Fig. S5). Considering this evidence, we characterised the expression of miR-193a-5p in vitro in a model system of human hepatocytes in the context of lipid accumulation as seen in NAFLD (Fig. 4). Hep G2 cells were loaded with fatty acids, and miR-193a-5p expression was quantified by qPCR. After a 24-h treatment with lipotoxic palmitic acid, miR- 193a-5p expression was decreased significantly relative to the control condition (p= 0.009). A less lipotoxic but more steatogenic lipid, oleic acid, 32 had a negligible effect relative to the control condition, and, also in line with a previous report, 32 there was no effect when the 2 lipids were combined. Discussion An unbiased profile of circulating miRNAs in NAFLD patients and controls has been generated using high-throughput sequencing techniques. Although other studies have identified some changes in serum miRNA levels in NAFLD, 17,33,34 our study is the largest of its kind –in terms of sample number and number of miRNAs analysed –spanning the full spectrum of the disease including cirrhosis. Although targeting only known miRNAs and unable to detect novel miRNAs, within these limitations we conducted a comprehensive and unbiased profile of miRNAs in NAFLD. Our sequencing approach revealed hundreds of miRNAs showing different levels at every NAFLD fibrosis stage relative to the controls, suggesting that these levels might correlate with, and therefore potentially predict, disease progression. We also saw numerous significant differences in miRNA levels within the entire NAFLD group compared with controls. Altered serum miRNA levels could be a useful diagnostic tool for NAFLD generally, but identifying such changes was not an aim of this study. By combining the NAFLD sequencing data with an independent qPCR replication, we have shown consistently that serum levels of miR-193a-5p can distinguish between mild and advanced disease activity and fibrosis. Our finding for miR-193a- 5p is in line with a recent report that performed miRNA sequencing of plasma samples from NAFLD cases but focussed on predominantly mild steatosis and fibrosis. 34 We also saw increased levels of miR-122-5p, which was generally in line with previous reports, 16,17,35 apart from a failure to detect significant differences with increasing fibrosis severity. This suggests that miR-193a-5p is a more sensitive biomarker of NAFLD progression than miR-122-5p. In addition to miR-193a-5p and miR-122-5p, we detected several other miRNAs that show differences in levels as NAFLD progresses, although the effect was not as significant as that for miR-193a-5p and overall levels of these miRNAs were lower. These additional significant miRNAs included miR-378d and miR-378e, which both increased in NASH and advanced fibrosis. This finding is in line with a recent sequencing study by Zhang et al., 34 who, while reporting that these 2 miRNAs were present at higher levels in plasma from NAFLD cases compared with controls and showed a significant relationship with extent of fibrosis, also found lower levels in plasma compared with miR-193a-5p, in both NAFLD cases and controls. We also saw significantly raised levels of miR-4484 in NASH compared with NAFL. The previous sequencing study reported levels of this miRNA increased in severe steatosis but did not include data on inflammation. 34 Our sequencing analysis did not detect changes in levels of miR-34a, another previously reported miRNA that increased in NAFLD, 19 but we were able to confirm previous data for this relatively low-abundance miRNA as a significant marker for NAFLD progression by use of a highly sensitive qPCR assay. Indicating its potential usefulness as a biomarker, serum miR-193a-5p achieved high AUROC values in our study. There is increasing evidence that at least a proportion of circulating miRNAs is encapsulated within extracellular vesicles in NAFLD. 36 The sequencing approach we used detects all miRNAs within serum without distinguishing between those present in vesicles and those circulating outside vesicles that are possibly protein-bound. There is evidence from other studies that levels of miR- 193a-5p are decreased in liver tissue in fibrosis 37 and in hepatocellular carcinoma 38 and that hepatic miR-193a-5p levels exhibit an inverse correlation with serum levels 37 similar to that reported for other miRNAs including miR-122-5p. 17,39 This would also be broadly consistent with our observations in Hep G2 cells where proapoptotic palmitic acid 32 decreases miR- 193a levels but steatogenic oleic acid, either alone or in combination with palmitic acid, has no significant effect. The miR- 193a target genes, found to show altered expression during NAFLD in our transcriptomic analysis, all increased in expression, which is typical of genes regulated by miRNA with a decreased miRNA level facilitating increased mRNA levels. In a previous study, miR-193a-5p has been shown to act within a network of miRNAs to modify the transforming growth factor- Research article 8JHEP Reports 2022 vol. 4 j100409