Serum coding and non-coding RNAs as biomarkers of NAFLD and fibrosis severity

Stefania Di Mauro, Alessandra Scamporrino, Salvatore Petta, Francesca Urbano, Agnese Filippello, Marco Ragusa, Maria T. Di Martino, Francesca Scionti, Stefania Grimaudo, Rosaria M. Pipitone, Graziella Privitera, Antonino Di Pino, Roberto Scicali, Luca Valenti, Paola Dongiovanni, Anna Fracanzani, Agata M. Rabuazzo, Antonio Craxì, Michele Purrello, Francesco PurrelloSalvatore Piro

Research output: Contribution to journalArticle

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Abstract

BACKGROUND & AIMS: In 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: We 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.

Original languageEnglish
Pages (from-to)1742-1754
Number of pages13
JournalLiver international : official journal of the International Association for the Study of the Liver
Volume39
Issue number9
DOIs
Publication statusPublished - Sep 1 2019

Fingerprint

Untranslated RNA
Liver Cirrhosis
Biomarkers
Palmitates
Long Noncoding RNA
Serum
Fibrosis
Fatty Liver
Up-Regulation
Oleic Acid
Biopsy
Messenger RNA
Area Under Curve
Non-alcoholic Fatty Liver Disease
Liver
Hep G2 Cells
Gene Expression Profiling
Disease Progression
Real-Time Polymerase Chain Reaction
Cohort Studies

Keywords

  • fibrosis
  • liquid-biopsy
  • NAFLD
  • NASH
  • RNAs

ASJC Scopus subject areas

  • Hepatology

Cite this

Serum coding and non-coding RNAs as biomarkers of NAFLD and fibrosis severity. / Di Mauro, Stefania; Scamporrino, Alessandra; Petta, Salvatore; Urbano, Francesca; Filippello, Agnese; Ragusa, Marco; Di Martino, Maria T.; Scionti, Francesca; Grimaudo, Stefania; Pipitone, Rosaria M.; Privitera, Graziella; Di Pino, Antonino; Scicali, Roberto; Valenti, Luca; Dongiovanni, Paola; Fracanzani, Anna; Rabuazzo, Agata M.; Craxì, Antonio; Purrello, Michele; Purrello, Francesco; Piro, Salvatore.

In: Liver international : official journal of the International Association for the Study of the Liver, Vol. 39, No. 9, 01.09.2019, p. 1742-1754.

Research output: Contribution to journalArticle

Di Mauro, S, Scamporrino, A, Petta, S, Urbano, F, Filippello, A, Ragusa, M, Di Martino, MT, Scionti, F, Grimaudo, S, Pipitone, RM, Privitera, G, Di Pino, A, Scicali, R, Valenti, L, Dongiovanni, P, Fracanzani, A, Rabuazzo, AM, Craxì, A, Purrello, M, Purrello, F & Piro, S 2019, 'Serum coding and non-coding RNAs as biomarkers of NAFLD and fibrosis severity', Liver international : official journal of the International Association for the Study of the Liver, vol. 39, no. 9, pp. 1742-1754. https://doi.org/10.1111/liv.14167
Di Mauro, Stefania ; Scamporrino, Alessandra ; Petta, Salvatore ; Urbano, Francesca ; Filippello, Agnese ; Ragusa, Marco ; Di Martino, Maria T. ; Scionti, Francesca ; Grimaudo, Stefania ; Pipitone, Rosaria M. ; Privitera, Graziella ; Di Pino, Antonino ; Scicali, Roberto ; Valenti, Luca ; Dongiovanni, Paola ; Fracanzani, Anna ; Rabuazzo, Agata M. ; Craxì, Antonio ; Purrello, Michele ; Purrello, Francesco ; Piro, Salvatore. / Serum coding and non-coding RNAs as biomarkers of NAFLD and fibrosis severity. In: Liver international : official journal of the International Association for the Study of the Liver. 2019 ; Vol. 39, No. 9. pp. 1742-1754.
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abstract = "BACKGROUND & AIMS: In 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: We 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.",
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author = "{Di Mauro}, Stefania and Alessandra Scamporrino and Salvatore Petta and Francesca Urbano and Agnese Filippello and Marco Ragusa and {Di Martino}, {Maria T.} and Francesca Scionti and Stefania Grimaudo and Pipitone, {Rosaria M.} and Graziella Privitera and {Di Pino}, Antonino and Roberto Scicali and Luca Valenti and Paola Dongiovanni and Anna Fracanzani and Rabuazzo, {Agata M.} and Antonio Crax{\`i} and Michele Purrello and Francesco Purrello and Salvatore Piro",
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TY - JOUR

T1 - Serum coding and non-coding RNAs as biomarkers of NAFLD and fibrosis severity

AU - Di Mauro, Stefania

AU - Scamporrino, Alessandra

AU - Petta, Salvatore

AU - Urbano, Francesca

AU - Filippello, Agnese

AU - Ragusa, Marco

AU - Di Martino, Maria T.

AU - Scionti, Francesca

AU - Grimaudo, Stefania

AU - Pipitone, Rosaria M.

AU - Privitera, Graziella

AU - Di Pino, Antonino

AU - Scicali, Roberto

AU - Valenti, Luca

AU - Dongiovanni, Paola

AU - Fracanzani, Anna

AU - Rabuazzo, Agata M.

AU - Craxì, Antonio

AU - Purrello, Michele

AU - Purrello, Francesco

AU - Piro, Salvatore

PY - 2019/9/1

Y1 - 2019/9/1

N2 - BACKGROUND & AIMS: In 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: We 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.

AB - BACKGROUND & AIMS: In 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: We 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.

KW - fibrosis

KW - liquid-biopsy

KW - NAFLD

KW - NASH

KW - RNAs

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