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Pre-Diabetes Linked Microrna Mir-193B-3P Targets PPARGC1A and Increases Lipid Accumulation in Hepatocytes: Potential Biomarker of Fatty Liver Disease

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Pre-Diabetes Linked Microrna Mir-193B-3P Targets PPARGC1A and Increases Lipid Accumulation in Hepatocytes: Potential Biomarker of Fatty Liver Disease bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427820; this version posted January 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Pre-diabetes linked microRNA miR-193b-3p targets PPARGC1A and increases lipid accumulation in hepatocytes: potential biomarker of fatty liver disease Inês Guerra Mollet1,*, Maria Paula Macedo1,2,3,* 1Chronic Diseases Research Centre (CEDOC), NOVA Medical School - Universidade NOVA de Lisboa (NMS-UNL), 1150-082 Lisbon, Portugal 2Associação Protectora dos Diabéticos de Portugal - Education Research Center (APDP-ERC), 1250-203 Lisbon, Portugal 3Department of Medical Sciences, Universidade de Aveiro, 3810-193 Aveiro, Portugal *Corresponding authors: Inês Guerra Mollet or Maria Paula Macedo Emails: [email protected] or [email protected] Running title: miR-193b causes lipid accumulation in hepatocytes Keywords microRNA (miRNA), hsa-miR-193b-3p, peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α)(PPARGC1A), hepatocyte, lipid droplet, fatty acid metabolism, liver metabolism, fatty liver, diabetes, metabolic syndrome Suggested journal section: Molecular Bases of Disease 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427820; this version posted January 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract mRNA into protein and directing the mRNA to Distinct plasma microRNA profiles associate nonsense mediated decay (2). We hypothesize with different disease features. Elevated plasma that particular microRNAs detected in microRNA hsa-miR-193b-3p has been reported circulation in early disease states can be used as in patients with pre-diabetes where early liver biomarkers of underlying dysmetabolism and dysmetabolism plays a crucial role. MicroRNA also that the targets (3) of each microRNA can target databases revealed that hsa-miR-193b-3p provide specific crucial organ specific could potentially target PPARGC1A, a master pathological information that could be used to switch transcriptional co-activator that inform early clinical intervention. Several orchestrates the expression of genes involved in circulating miRNAs have been reported as being multiple metabolic pathways. In this study we altered in plasma from patients with metabolic evaluated the effects of hsa-miR-193b-3p in the disease including pre-diabetes, however, the human HepG2 hepatocyte cell line. We show that specific mechanisms through which they hsa-miR-193b-3p targets PPARGC1A/PGC1a function remain unexplored (4). mRNA 3'UTR and reduces its expression. Pre-diabetes, diagnosed as impaired Overexpression of hsa-miR-193b-3p under fasting glucose and/or impaired glucose hyperglycemia resulted in increased tolerance, is considered an early sign of accumulation of intracellular lipid droplets; and metabolic syndrome (5). NAFLD/MAFLD quantitative real-time RT-PCR of a panel of 24 (Nonalcoholic fatty Liver Disease/Metabolic genes revealed significant disruption in the Associated fatty Liver Disease) (6) is a precursor expression of genes coordinating glucose uptake, of metabolic syndrome (7, 8). NAFLD/MAFLD glycolysis, pyruvate metabolism, fatty acid is a condition characterized by a fatty liver, synthesis, fatty acid oxidation, triglyceride which can originate complications, including synthesis, VLDL secretion, insulin signalling, cirrhosis, liver failure, liver cancer, and and mitochondrial biogenesis, dynamics and cardiometabolic health problems that encompass function. These results provide in vitro evidence dysmetabolism. Thus, analyzing the effects of that microRNA hsa-miR-193b-3p downregulates pre-diabetes associated circulating microRNA PPARGC1A/PGC1a, disrupts downstream targets in liver cells is an appropriate premise to metabolic pathways and leads to the identify early intervention targets that may have accumulation of intracellular lipids in potentially important clinical application in hepatocytes. We propose that microRNA hsa- diagnosis, prevention and treatment, paving the miR-193b-3p may have potential as a clinically way for precision medicine. Among these is relevant plasma biomarker for metabolic microRNA hsa-miR-193b-3p, which is associated fatty liver disease in a pre-diabetic overexpressed in plasma of patients with dysglycemic context. impaired fasting glucose and glucose intolerance and which tends to normalize upon exercise Introduction intervention in humans (4, 9) but for which no MicroRNAs are small non-protein coding 20-22 data exists on a precise mechanism or relevant nucleotide long RNAs produced by cells of targeting. various tissues that can be actively released into In this study we demonstrate that hsa- the bloodstream in vesicles called exosomes and miR-193b-3p targets the expression of delivered to cells in the same or other tissues in PPARGC1A mRNA and increases lipid the organism (1). MicroRNAs target protein accumulation in human hepatocyte derived cells. expression essentially by binding to the 3' PPARGC1A codes for PGC-1a a central co- untranslated region (3'UTR) of messenger RNA activator of transcriptional cascades that regulate (mRNA) thereby preventing translation of the several interconnected pathways including 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427820; this version posted January 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. glucose metabolism (10), lipid metabolism (11, Results 12), inflammation, mitochondrial function and MicroRNA hsa-miR-193b-3p targets the 3'UTR oxidative stress (13). PGC-1a functions by of PPARGC1A mRNA and downregulates interacting with the nuclear peroxisome PPARGC1A expression in HepG2 cells. To proliferator-activated receptors (PPARs) evaluate potential mRNA targets of microRNA activating them to bind DNA in complex with hsa-miR-193b-3p we performed a bioinformatic retinoid X receptor (RXR) (14), on the promoter analysis of conserved predicted targets of hsa- of numerous genes central to metabolic miR-193b-3p from the online database regulation, increasing transcription of specific TargetScan 7.1 (3) using the DAVID genes and decreasing transcription of others. The Bioinformatics Resources 6.8 (17, 18) and peroxisome proliferator activated receptors clustering tools for functional Gene Ontology (PPARs) are nuclear receptors that play key roles Annotations (19). Of the 283 predicted in the regulation of lipid and glucose metabolism, conserved mRNA targets of microRNA hsa- inflammation, cellular growth, and miR-193b-3p we found the top gene ontology differentiation. The receptors bind and are cluster was composed of 43 genes involved in activated by a broad range of fatty acids and fatty gene transcription. from this list PPRAGC1A acid derivatives serving as major transcriptional stood out as the gene with the most consistently sensors of fatty acids (14). We therefore also conserved microRNA hsa-miR-193b-3p target analyzed the expression of several metabolic site involved in energy metabolism. Peroxisome master-switch genes downstream of hsa-miR- Proliferator-Activated Receptor Gamma 193b-3p downregulation of Coactivator 1-Alpha (PPARGC1A) codes for PPRAGC1A/PGC1a to analyze downstream PGC-1α, a transcriptional coactivator of pathway regulation. Glucose and lipid numerous genes involved in energy metabolism. metabolism depend on the ability of When hsa-miR-193b-3p was overexpressed in mitochondria to generate energy in cells (15), HepG2 cells for 72 hours using a mimic, we therefore we also looked at hepatic mitochondrial observed robust downregulation of PPARGC1A dysfunction which plays a central role in the mRNA expression at basal glucose concentration pathogenesis of insulin resistance in obesity, pre- (5mM glucose, figure 1A), under hyperglycemia diabetes and NAfLD/MAfLD (16). (20 mM glucose, figure 1B), under We sought to investigate whether hyperinsulinemia (5mM glucose and final 24h elevated levels of microRNA hsa-miR-193b-3p 10nM insulin, figure 1C) and under in hepatocytes could interfere with hepatic cell hyperglycemia/hyperinsulinemia (20mM function in vitro under conditions that mimic pre- glucose and final 24h 10nM insulin, figure 1D). diabetes. We evaluated pre-diabetes associated We validated the predicted PPARGC1A 3'UTR microRNA hsa-miR-193b-3p predicted target target site for miR-193b-3p by cloning a PPARGC1A (PGC1a), hepatic lipid content and "match", or a "mismatch" control (Figure 1E), direct or indirect effects on expression of genes into the 3'UTR of the firefly luciferase gene in regulating cellular energy metabolism, in human the pmirGLO Dual-Luciferase miRNA Target hepatocyte derived cells. The objective was to Expression Vector (Figure 1F), followed by investigate which metabolic pathways dual-glow luciferase assay. Expression of firefly microRNA hsa-miR-193b-3 interfered with so as luciferase from the "match" clone was to gage the potential of hsa-miR-193b-3p as an significantly downregulated in HepG2 cells early plasma biomarker of specific liver when the hsa-miR-193b-3p mimic was dysmetabolism to support precision medicine in overexpressed and cells were transfected with early stages. pmirGLO match clone (Figure 1G). These results support our posit that microRNA hsa-miR-193b- 3 bioRxiv preprint
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