Roles for the long non-coding RNAPax6os1/PAX6-AS1in pancreatic beta cell identity and function
Authors: Lopez-Noriega Livia , Callingham Rebecca , Martinez-Sánchez Aida , Nawaz Sameena , Pizza Grazia , Haberman Nejc , Cvetesic Nevena , Nguyen-Tu Marie-Sophie , Lenhard Boris , Marchetti Piero , Piemonti Lorenzo , de Koning Eelco , Shapiro A. M. James , Johnson Paul R. , Leclerc Isabelle , Hastoy Benoit , Gauthier Benoit R. , Pullen Timothy J. , Rutter Guy A. 
Published: July 2020

AbstractAim/HypothesisLong non-coding RNAs (lncRNAs) are emerging as crucial regulators of beta cell development and function. Here, we investigate roles for an antisense lncRNA expressed from thePax6locus (annotated asPax6os1in mice andPAX6-AS1in humans) in beta cell identity and functionality.MethodsPax6os1expression was silenced in MIN6 cells using siRNAs and changes in gene expression were determined by RNA sequencing or qRT-PCR. Mice inactivated forPax6os1and humanPAX6-AS1-null EndoC-βH1 cells, were generated using CRISPR/Cas9 technology. Human islets were infected with lentiviral vectors bearing a targeted shRNA orPAX6-AS1, which were used to silence or overexpress, respectively, the lncRNA. RNA sequencing or RT-qPCR were used to measure transcriptomic changes and RNA pulldown in mice and human cells followed by mass spectrometry/western blot were performed to explore RNA protein interactions.ResultsPax6os1/PAX6-AS1expression was upregulated at high glucose concentrations in derived beta cell lines as well as in mouse and human islets, and in pancreatic islets isolated from mice fed a high fat diet (n=6, p=0.003) and patients with type 2 diabetes (n=11-5, p<0.01). Silencing or deletion ofPax6os1/PAX6-AS1in MIN6 or EndoC-βH1cells increased the expression of several β-cell signature genes, includingPDX1andINS. Female, but not male,Pax6os1null mice fed a high fat diet showed slightly enhanced glucose clearance. ShRNA-mediated silencing ofPAX6-AS1in human islets robustly increasedINSmRNA, enhanced glucose-stimulated insulin secretion and calcium dynamics, while overexpression of the lncRNA exerted opposing effects.Pax6os1/AS-1interacted with histones H3 and H4 in mouse and human cells, indicating a possible role for this lncRNA in histone modifications in both species.ConclusionsIncreased expression ofPAX6-AS1at high glucose levels may impair beta cell functionality and thus contribute to the development of type 2 diabetes. Thus, targetingPAX6-AS1may provide a promising strategy to enhance insulin secretion and improve glucose homeostasis in this disease.Research in contextWhat is already known about the subject?Long non-coding RNAs (lncRNAs) are crucial components of the pancreatic islet regulome, whose misexpression may contribute to the development of diabetes.What is the key question?Is the lncRNAPax6os1/PAX6-AS1involved in beta cell functionality and type 2 diabetes?What are the new findings?The expression ofPax6os1/PAX6-AS1is upregulated in mice fed a high fat diet and in pancreatic islets from type 2 diabetes donors.Overexpression ofPAX6-AS1in human pancreatic islets reduces insulin expression, glucose stimulated secretion and intracellular calcium dynamics.SilencingPAX6-AS1in human pancreatic islets upregulates insulin expression, enhances glucose stimulated insulin secretion and increases intracellular calcium dynamics.How may this impact the clinic in the foreseeable future?Understanding the genetic factors induced by high glucose/obesity involved in beta cell dysfunction is crucial for the development of new therapies to treat T2D.