TITOLO DEL PROGETTO
Pancreatic islet microRNAs as therapeutic target in type 2 diebetes
Prof. Francesco Dotta
Inizio: Settembre 2010 – Conclusione: Settembre 2013
La Regione Toscana, nell'ambito del Programma per la Ricerca Regionale in Materia di Salute 2009, ha finanziato il progetto "Pancreatic islet microRNAs as therapeutic target in type 2 diebetes", che è stato condotto presso i laboratori della Fondazione Umberto Di Mario ONLUS presso la Fondazione Toscana Life Sciences, sotto la direzione scientifica del Prof. Francesco Dotta.
MicroRNAs (miRNAs or miRs) are a recently identified class of small cellular RNAs, whose function is to pair the mRNAs of protein-coding genes with subsequent transcriptional and post-transcriptional regulation of gene expression. MicroRNAs have emerged as important regulatory factors involved in developmental processes, such as neural progenitor cell growth and differentiation and their altered expression has been observed in a large number of malignancies. In addition, increasing evidence has shown that miRNAs are involved in the development of endocrine pancreas as well as in the regulation of insulin secretion and insulin signalling. In a series of preliminary experiments, we discovered a differentially miRNA expression profiling in type 2 diabetic vs control human pancreatic islets which includes a major hyperexpression of miR-124, whose silencing resulted in increased insulin secretion and expression of genes involved in beta cell function and development, leading to the hypothesis that an altered miRNA expression may contribute to beta cell defects in type 2 diabetes. Moreover, we reported the first evidence that miR-124 directly targets molecules involved in insulin secretion and in the insulin signalling pathway in human cells. In the present project, we plan to dissect the impact of modulating the expression of miRNAs of interest (identified in our preliminary study) on insulin-secreting beta cells, both in terms of cell survival and proliferation and in terms of islet cell function. Experiments will be performed in 3 different settings:
- Insulin or glucagon secreting cell lines (beta-TC, MIN6, INS1E, alpha-TC)
- In vitro cultured isolated mouse or human pancreatic islets and LCM-purified islet alpha- and beta-cells from pancreatic frozen sections obtained from non-diabetic or type 2 diabetic organ donors
- Experimental mouse models of diabetes mellitus.
In all above mentioned settings, expression of miRNAs of interest (miR-124; miR-187; miR-138; miR-142; miR-184; miR-31; miR-213; miR-337) will be inhibited (by RNA silencing) or overexpressed and the impact of such modulation will be evaluated on: islet cell proliferation and survival; insulin secretion; glucagon secretion; expression of target genes involved in molecular pathways of interest (e.g. insulin signalling, cell cycle, apoptosis). In vivo modulation of miRNA expression in mouse models of diabetes will allow to validate in vivo the results obtained in vitro on cell lines and on isolated pancreatic islets.
We have identified a series of microRNAs and their predicted target genes whose expression is modulated during the expansion and re-differentiation of human islet derived beta cell precursors. In addition, we have started to identify their putative target genes with particular reference to those encoding for molecules of potential interest in pancreatic beta cell biology. We also moved to the analysis of islet samples from type 2 diabetic donors, uncovering an islet "regenerative defect" characterized by impaired proliferation and defective properties in terms of re-differentiation into a pancreatic endocrine phenotype.