Monika Gjorgijeva Ducros, Hjelt Grant Holder 2022, University of Geneva. Background MicroRNAs (miRNAs) are critical gene expression regulators involved in mRNA decay or translation inhibition. MiRNAs play an important role in various physiological processes and therefore, deregulation of their expression/activity has been associated with the development of metabolic disorders. Obesity and the metabolic syndrome represent key etiological conditions that predispose to the development of insulin resistance (IR), Type 2 Diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Increasing evidence indicate that miRNA deregulation contributes to the development of these diseases. In this context, our recent findings highlighted a strong induction of miR-149 in the liver of various models of IR, T2D and NAFLD, suggesting an important role of this miRNA in these metabolic disorders. Hypothesis Based on our preliminary results, we hypothesize that the increase in hepatic mir-149 in IR/T2D/NAFLD conditions can favor these pathologies. We will therefore investigate i) the pathophysiological role and pre-clinical relevance of miR-149 upregulation in IR/T2D/NAFLD and ii) which miR-149 target genes are involved in this process. Methods To investigate the role of miR-149 in IR/T2D/NAFLD, we are using human liver organoids (HLOs). HLOs are obtained by inducing differentiation of human progenitor cells into different hepatic cell types (hepatocytes, Kuppfer cells, stellate cells) that form functional structures. These organoids respond to insulin stimulation in the same manner as human liver. Moreover, they develop hepatic steatosis under high-fat/high sugar conditions and can undergo inflammation when stimulated with cytokines. Finally, HLOs are an extremely relevant experimental model as they allow us to avoid animal experimentation protocols. Therefore, we will modulate the expression of miR-149 in HLOs via synthetic nucleotides and/ or viral vectors and we will analyze the molecular responses in HLO under various metabolic / inflammatory stimuli. We will further identify miR-149 target genes involved in IR/T2D/NAFLD and we will validate their relevance in public human datasets. Results Our preliminary data suggest a pertinent role for miR-149 upregulation in the induction of steatosis in vitro in currently used hepatic cell lines, as well as in HLO. We have also observed that alteration of miR-149 levels has a striking effect on hepatic glucose and lipid metabolism, implying a functional role for this miRNA in IR/T2D/NAFLD. Conclusion This project should allow us to better understand the role of miR-149 in hepatic IR, and more generally in obesity-associated disorders of the hepatic lipid/glucose metabolism. We will identify novel target genes of miR-149 contributing to IR/T2D/NAFLD and the fine-tuning metabolic regulation in the liver in pathophysiological conditions. Our investigations should provide key evidence and proof-of-concept about the potential of miR-149 and its targets as new biomarkers for IR/T2D as well as the therapeutic potential of targeting this miRNA to counteract and/or to alleviate IR/T2D development. Importance Modulation of miR-149 represents a promising therapeutic strategy by targeting numerous genes at once. Therefore, miR-149 modulation could represent a multi-targeting approach relevant and pertinent for multifactorial disorders such as obesity, IR/T2D/NAFLD. A significant and innovative aspect of this proposal is the development and use of genetically engineered functional HLOs in which hepatic steatosis, inflammation and IR can be reproduced. HLOs have the potential of replacing animal experimentation, thereby alleviating important ethical issues related to the use of laboratory mice for pre-clinical research.