Roles for the long non-coding RNA Pax6os1/PAX6-AS1 in pancreatic beta cell identity and function

Aim/Hypothesis Long 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 the Pax6 locus (annotated as Pax6os1 in mice and PAX6-AS1 in humans) in beta cell identity and functionality. Methods Pax6os1 expression was silenced in MIN6 cells using siRNAs and changes in gene expression were determined by RNA sequencing or qRT-PCR. Mice inactivated for Pax6os1 and human PAX6-AS1 -null EndoC-βH1 cells, were generated using CRISPR/Cas9 technology. Human islets were infected with lentiviral vectors bearing a targeted shRNA or PAX6-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. Results Pax6os1/PAX6-AS1 expression 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 of Pax6os1 / PAX6-AS1 in MIN6 or EndoC-βH1cells increased the expression of several β-cell signature genes, including PDX1 and INS . Female, but not male, Pax6os1 null mice fed a high fat diet showed slightly enhanced glucose clearance. ShRNA-mediated silencing of PAX6-AS1 in human islets robustly increased INS mRNA, enhanced glucose-stimulated insulin secretion and calcium dynamics, while overexpression of the lncRNA exerted opposing effects. Pax6os1/AS-1 interacted with histones H3 and H4 in mouse and human cells, indicating a possible role for this lncRNA in histone modifications in both species. Conclusions Increased expression of PAX6-AS1 at high glucose levels may impair beta cell functionality and thus contribute to the development of type 2 diabetes. Thus, targeting PAX6-AS1 may provide a promising strategy to enhance insulin secretion and improve glucose homeostasis in this disease. What 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 lncRNA Pax6os1/PAX6-AS1 involved in beta cell functionality and type 2 diabetes? What are the new findings? The expression of Pax6os1/PAX6-AS1 is upregulated in mice fed a high fat diet and in pancreatic islets from type 2 diabetes donors. Overexpression of PAX6-AS1 in human pancreatic islets reduces insulin expression, glucose stimulated secretion and intracellular calcium dynamics. Silencing PAX6-AS1 in 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. ### Competing Interest Statement GR has received grant funding from Sun Pharmaceuticals and Servier and is a consultant for Sun Pharmaceuticals. * EDU : 5-ethynyl-2’-deoxyuridine GSIS : glucose-stimulated insulin secretion; HbA1c : glycated haemoglobin IPGTT : intraperitoneal glucose tolerance test HFD : high fat diet KO : Knockout LncRNA : Long non-coding RNA MTT : 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide RNASeq : Massive parallel RNASequencing siRNA : small interfering RNA STD : Standard diet T2D : Type 2 Diabetes