AMPK‐regulated miRNAs in pancreatic β‐cells: underlying molecular mechanisms and high throughput target identification

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that silence gene expression post-transcriptionally and play a key role in the development of Type 2 Diabetes (T2D). In pancreatic β‐cells, miRNAs control glucose‐induced insulin secretion and content and their expression is altered in T2D and hyperglycaemia through unknown molecular mechanisms. MiR‐184, an important regulator of insulin secretion, and miR‐125b, whose role in β‐cells is still unknown, are both regulated by glucose through AMP‐activated protein kinase (AMPK), a nutrient sensor essential for cellular energy homeostasis and suggested target for anti-diabetes drugs. With this thesis we aimed to identify the mechanisms linked to AMPK-mediated transcription regulation of miR-184 and miR-125b in β-cells. We also aimed to elucidate miR-125b function in β-cells through high-throughput identification of its direct targets. Finally, we aimed to develop a high‐throughput method (AGO2 eiCLIP-seq) to identify all specific miRNA‐mRNA interactions in human β-cells. Our results suggest that AMPK mediates glucose-dependent miR-184 expression regulation independently of CTCF and that miR-184 TSS is located ~121 Kb upstream of MIR184 in a less accessible region in LKB1KO islets, suggesting a positive regulatory role in miR-184 transcription for AMPK. We also found that miR-125b expression is negatively regulated by SMAD3 and that the AMPK-mediated down-regulation of miR-125b might occur independently of SMAD3. Importantly, we identified miR-125b direct targets in β‐cells and found that miR-125b overexpression results in abnormally enlarged lysosomes that could contribute to impaired nutrient signalling and/or insulin biosynthesis. Finally, I optimized the usage of enhanced single-nucleotide resolution UV-crosslinking and immunoprecipitation (eiCLIP) against Argonaute 2 (AGO2 – an essential miRISC component) in EndoC-βH1 human β-cells, which will support the precise mapping of the entire β‐cell miRNA targetome. My findings provide new insights into the regulation and role of β-cell miRNAs and may contribute to the identification of future therapeutic targets for the treatment of T2D.