Agonist of RORA Attenuates Nonalcoholic Fatty Liver Progression in Mice via Up-regulation of MicroRNA 122

Chofit Chai, Bryan Cox, Dayana Yaish, Devora Gross, Nofar Rosenberg, Franck Amblard, Zohar Shemuelian, Maytal Gefen, Amit Korach, Oren Tirosh, Tali Lanton, Henrike Link, Joseph Tam, Anna Permyakova, Gunes Ozhan, Jonathan Citrin, Haixing Liao, Mirna Tannous, Michal Hahn, Jonathan AxelrodEnara Arretxe, Cristina Alonso, Ibon Martinez-Arranz, Pablo Ortiz Betés, Rifaat Safadi, Ahmad Salhab, Johnny Amer, Zahira Tber, Seema Mengshetti, Hilla Giladi, Raymond F. Schinazi*, Eithan Galun

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

Background & Aims: Development of nonalcoholic steatohepatitis (NASH) is associated with reductions in hepatic microRNA122 (MIR122); the RAR related orphan receptor A (RORA) promotes expression of MIR122. Increasing expression of RORA in livers of mice increases expression of MIR122 and reduces lipotoxicity. We investigated the effects of a RORA agonist in mouse models of NASH. Methods: We screened a chemical library to identify agonists of RORA and tested their effects on a human hepatocellular carcinoma cell line (Huh7). C57BL/6 mice were fed a chow or high-fat diet (HFD) for 4 weeks to induce fatty liver. Mice were given hydrodynamic tail vein injections of a MIR122 antagonist (antagomiR-122) or a control antagomiR once each week for 3 weeks while still on the HFD or chow diet, or intraperitoneal injections of the RORA agonist RS-2982 or vehicle, twice each week for 3 weeks. Livers, gonad white adipose, and skeletal muscle were collected and analyzed by reverse-transcription polymerase chain reaction, histology, and immunohistochemistry. A separate group of mice were fed an atherogenic diet, with or without injections of RS-2982 for 3 weeks; livers were analyzed by immunohistochemistry, and plasma was analyzed for levels of aminotransferases. We analyzed data from liver tissues from patients with NASH included in the RNA-sequencing databases GSE33814 and GSE89632. Results: Injection of mice with antagomiR-122 significantly reduced levels of MIR122 in plasma, liver, and white adipose tissue; in mice on an HFD, antagomiR-122 injections increased fat droplets and total triglyceride content in liver and reduced β-oxidation and energy expenditure, resulting in significantly more weight gain than in mice given the control microRNA. We identified RS-2982 as an agonist of RORA and found it to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed an HFD or atherogenic diet, injections of RS-2982 increased hepatic levels of MIR122 precursors and reduced hepatic synthesis of triglycerides by reducing expression of biosynthesis enzymes. In these mice, RS-2982 significantly reduced hepatic lipotoxicity, reduced liver fibrosis, increased insulin resistance, and reduced body weight compared with mice injected with vehicle. Patients who underwent cardiovascular surgery had increased levels of plasma MIR122 compared to its levels before surgery; increased expression of plasma MIR122 was associated with increased levels of plasma free fatty acids and levels of RORA. Conclusions: We identified the compound RS-2982 as an agonist of RORA that increases expression of MIR122 in cell lines and livers of mice. Mice fed an HFD or atherogenic diet given injections of RS-2982 had reduced hepatic lipotoxicity, liver fibrosis, and body weight compared with mice given the vehicle. Agonists of RORA might be developed for treatment of NASH.

Original languageAmerican English
Pages (from-to)999-1014.e9
JournalGastroenterology
Volume159
Issue number3
DOIs
StatePublished - Sep 2020

Bibliographical note

Funding Information:
Funding This work was supported by an ERC advance (GA no. 786575) RxmiRcanceR (to Eithan Galun), Deutsche Forschungsgemeinschaft SFB841 project C3 (to Eithan Galun), National Institutes of Health CA197081-02 (to Eithan Galun), MOST (to Eithan Galun), ISF collaboration with Canada (2473/2017) (to Eithan Galun), personal ISF (486/2017) (to Eithan Galun), ICORE-ISF (41/2011) (to Eithan Galun), DKFZ-MOST (to Eithan Galunthe Robert H. Benson Living Trust, and Selma Kron Foundation to student fellowships.

Funding Information:
The authors wish to thank Yuval Nevo, Sharona Elgavish, and the Hadar Benyamini Bioinformatics Unit of the Israeli Centers of Research Excellence (I-CORE) Computation Center, The Hebrew University and Hadassah Hebrew University Hospital, Jerusalem, for the bioinformatic analysis conducted during the project; Devorah Olam for the histologic analysis conducted in this project; J?rg Heeren and Anna Worthmann for collaboration and discussion; and Israel Voldavsky, who gave us the protocol for the use of heparinase for eliminating heparin from the human samples. Chofit Chai, PhD (Investigation: Lead); Bryan Cox, PhD (Investigation: Supporting); Dayana Yaish, PhD student (Investigation: Supporting); Devora Gross, MSc student (Investigation: Supporting); Nofar Rosenberg, PhD student (Investigation: Supporting); Franck Amblard, PhD (Investigation: Supporting); Zohar Shemuelian, MSc student (Investigation: Supporting); Maytal Gefen, PhD student (Investigation: Supporting); Amit Korach, MD (Investigation: Supporting); Oren Tirosh, PhD (Investigation: Supporting); Tali Lanton, PhD (Investigation: Supporting); Henrike Link, MD student (Investigation: Supporting); Joseph Tam, Prof (Investigation: Supporting); Anna Permikov, PhD (Investigation: Supporting); Gunes Ozhan, MD (Investigation: Supporting); Jonathan Citrin, medical student (Investigation: Supporting); Haixing Liao, MD (Investigation: Supporting); Mirna Tannous, MSc student (Investigation: Supporting); Michal Hahn, PhD (Investigation: Supporting); Jonathan Axelrod, PhD (Investigation: Supporting); Enara Arretxe, PhD (Investigation: Supporting); Cristina Alonso, PhD (Investigation: Supporting); Ibon Martinez-Arranz, PhD (Investigation: Supporting); Pablo Ortiz Bet?s, PhD (Investigation: Supporting); Rifaat Safadi, MD (Investigation: Supporting); Ahmad Salhab, PhD student (Investigation: Supporting); Johnny Amer, PhD (Investigation: Supporting); Zahira Tber, PhD (Investigation: Supporting); Seema Mengshetti, PhD (Investigation: Supporting); Hilla Giladi, PhD (Investigation: Equal; Writing ? review & editing: Equal); Raymond F, Schinazi, Prof (Conceptualization: Equal; Investigation: Equal; Writing ? original draft: Equal); Eithan Galun, Prof (Conceptualization: Lead; Formal analysis: Equal; Funding acquisition: Lead; Project administration: Equal; Writing ? review & editing: Equal). Funding This work was supported by an ERC advance (GA no. 786575) RxmiRcanceR (to Eithan Galun), Deutsche Forschungsgemeinschaft SFB841 project C3 (to Eithan Galun), National Institutes of Health CA197081-02 (to Eithan Galun), MOST (to Eithan Galun), ISF collaboration with Canada (2473/2017) (to Eithan Galun), personal ISF (486/2017) (to Eithan Galun), ICORE-ISF (41/2011) (to Eithan Galun), DKFZ-MOST (to Eithan Galunthe Robert H. Benson Living Trust, and Selma Kron Foundation to student fellowships.

Publisher Copyright:
© 2020 AGA Institute

Keywords

  • Lipid
  • Metabolism
  • NAFLD
  • Obesity

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