miR-34a/SIRT1/p53 is suppressed by ursodeoxycholic acid in the rat liver and activated by disease severity in human non-alcoholic fatty liver disease.
OBJECTIVE
Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis (NASH). However, disease pathogenesis remains largely unknown. microRNA (miRNA or miR) expression has recently been reported to be altered in human NASH, and modulated by ursodeoxycholic acid (UDCA) in the rat liver. Here, we aimed at evaluating the miR-34a/Sirtuin 1(SIRT1)/p53 pro-apoptotic pathway in human NAFLD, and to elucidate its function and modulation by UDCA in the rat liver and primary rat hepatocytes.


METHODS
Liver biopsies were obtained from NAFLD morbid obese patients undergoing bariatric surgery. Rat livers were collected from animals fed a 0.4% UDCA diets. Primary rat hepatocytes were incubated with bile acids or free fatty acids (FFAs) and transfected with a specific miRNA-34a precursor and/or with a p53 overexpression plasmid. p53 transcriptional activity was assessed by ELISA and target reporter constructs.


RESULTS
miR-34a, apoptosis and acetylated p53 increased with disease severity, while SIRT1 diminished in the NAFLD liver. UDCA inhibited the miR-34a/SIRT1/p53 pathway in the rat liver in vivo and in primary rat hepatocytes. miR-34a overexpression confirmed its targeting by UDCA, which prevented miR-34a-dependent repression of SIRT1, p53 acetylation, and apoptosis. Augmented apoptosis by FFAs in miR-34a overexpressing cells was also inhibited by UDCA. Finally, p53 overexpression activated miR-34a/SIRT1/p53, which in turn was inhibited by UDCA, via decreased p53 transcriptional activity.


CONCLUSIONS
Our results support a link between liver cell apoptosis and miR-34a/SIRT1/p53 signaling, specifically modulated by UDCA, and NAFLD severity. Potential endogenous modulators of NAFLD pathogenesis may ultimately provide new tools for therapeutic intervention.
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal. ruieduardocastro@ff.ul.pt
Rui E
RE
Castro
Duarte M S
DM
Ferreira
Marta B
MB
Afonso
Pedro M
PM
Borralho
Mariana V
MV
Machado
Helena
H
Cortez-Pinto
Cecília M P
CM
Rodrigues
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001706:Biopsy; D005234:Fatty Liver / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D022781:Hepatocytes / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D065626:Non-alcoholic Fatty Liver Disease; D009767:Obesity, Morbid / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D061251:Primary Cell Culture; D051381:Rats; D017207:Rats, Sprague-Dawley; D012720:Severity of Illness Index; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D056564:Sirtuin 1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D014580:Ursodeoxycholic Acid / Q000378:metabolism* / Q000494:pharmacology
C522425:MIRN34 microRNA, human; C560944:MIRN34 microRNA, rat; D035683:MicroRNAs; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D014580:Ursodeoxycholic Acid; C447939:SIRT1 protein, human; C488368:Sirt1 protein, rat; D056564:Sirtuin 1