ESPE Abstracts (2018) 89 FC6.6

ESPE2018 Free Communications Fat, Metabolism and obesity (6 abstracts)

MicroRNA-141 Directly Targets and Inhibits Sirtuins 1 Gene Expression and Its Elevation in Obese Subjects is Responsible for Reduced Levels of Sirtuin 1 and the Subsequent Hepatic Steatosis and Insulin Resistance

Mitra Nourbakhsh a , Zeynab Yousefi a , Nikta Dadkhah Nikroo b , Mojtaba Malek c , Abdolreza Pazouki d & Somayye Mokhber d

aDepartment of Biochemistry, School of Medicine, Iran University of Medical Sciences, Tehran, Islamic Republic of Iran; bDepartment of Biology, Science and Research Branch, Islamic Azad University, Tehran, Islamic Republic of Iran; cResearch Center for Prevention of Cardiovascular Disease, Institute of Endocrinology and Metabolism, Iran University of Medical Sciences, Tehran, Islamic Republic of Iran; dMinimally Invasive Surgery Research Center, Iran University of Medical Sciences, Tehran, Islamic Republic of Iran

Introduction: Obesity increases the risk of various disorders including diabetes, non-alcoholic fatty liver disease (NAFLD) and cardiovascular disorders. MicroRNAs (miRNA) are single-stranded, non-coding oligonucleotides that regulate gene expression. Sirtuin 1 (SIRT1), a regulatory enzyme in metabolic homeostasis, is regulated by miRNAs. The aim of this study was to evaluate miR-141 in obesity and whether this miRNA can regulate SIRT1 expression.

Materials and methods: Plasma levels of miR-141 were measured by real-time PCR in samples from 50 obese subjects and 50 normal-weight control subjects after thorough clinical evaluation. Peripheral blood mononuclear cells were isolated and used for the assessment of SIRT1 expression after mRNA extraction and cDNA biosynthesis. Lipid profile, glucose and insulin as well as liver function tests were also measured in serum samples. Hepatic steatosis was evaluated by ultrasound. Targeting of SIRT1 by miR-141 were first evaluated by bioinformatic tools. Then luciferase reporter assay was used to confirm direct interaction of miR-141 with 3’-untraslated region (3’-UTR) of SIRT1 mRNA after co-transfection of HEK293 cells with miR-141 mimic and the vector containing the 3’-UTR sequence. Levels of SIRT1 protein and SIRT1 activity were determined by Western blotting and a fluorimetric method, respectively, after transfection of HepG2 liver cells by miR-141 mimic and inhibitor and their negative controls.

Results: miR-141 levels were increased while SIRT1 gene expression was decreased in obese subjects compared to normal subjects. SIRT1 expression was negatively correlated with miR-141 levels. High plasma miR-141 and low SIRT1 expression were associated with fat accumulation in liver as well as insulin resistance. The same alterations were observed in HepG2 cells after incubation with high concentrations of glucose and induction of intracellular lipid accumulation. Luciferase reporter assay together with bioinformatic evaluation showed that SIRT1 is a direct target of miR-141. The expression of miR-141 was up-regulated while SIRT1 was down-regulated as a result of transfection of HepG2 cells with miR-141. Additionally, the protein level of SIRT1 and its activity were decreased due to the over expression of miR-141.

Conclusion: Results showed that miR-141 could effectively suppress SIRT1 expression and inhibit its activity. Thus, increased miR141 level in obesity could be considered as a causative factor for the low expression level of the SIRT1 gene. Consequently, the decrease in the expression and activity of SIRT1 could contribute to the development of insulin resistance and NAFLD.

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