MicroRNA regulation of endothelial calcium signalling

The Ca2+ signalling pathway is fundamental for many endothelial functions including angiogenesis, regulation of blood vessel tone and barrier selectivity and must therefore be regulated appropriately. MicroRNAs are non-coding RNAs which can regulate the expression of multiple genes at the posttranscriptional level. The aim of this thesis was to investigate the possibility that microRNAs can regulate Ca2+ responses by targeting genes of the IP3-Ca2+ signalling pathway. The microRNA expression profile of primary bovine retinal microvascular endothelial cells (RMECs), a widely used in vitro model for studies of endothelial physiology and angiogenesis, was analysed by high throughput sequencing. Expression of genes involved in IP3-Ca2+ signalling was assessed by RT-PCR. MicroRNA-26a was selected for further analysis because it was highly expressed and predicted to target key genes of IP3-Ca2+ signalling expressed in RMECs, namely IP3 receptor type 1 (ITPRl) and phospholipase C beta 1 (PLCBl). Changes in ITPRl mRNA and protein following overexpression or knockdown of microRNA-26a were consistent with it being a target and luciferase assays confirmed a direct interaction. Uridine triphosphate induced a transient increase in intracellular calcium concentration ([Ca2+]i) measured by fura-2 Ca2+ microfluorimetry that was blocked by PLC and IP3 receptor inhibitors. Overexpression of microRNA-26a decreased this elevation in [Ca2+]i and knockdown of microRNA-26a increased the response. This effect is likely to be at least partially due to the regulation of ITPRl. Many other genes that are modulated by microRNA-26a and may mediate the functional responses in RMECs were detected by high throughput mRNA sequencing. Analysis of the functions associated with these genes supports a role for microRNA-26a in the regulation of vascular physiology. The results suggest that microRNA-26a can modulate the Ca2+ responses of RMECs by targeting the IP3-Ca2+ signalling pathway. Further studies are now warranted to determine the functional significance of microRNA-26a in both retinal microvascular endothelial cell physiology and pathophysiology.EThOS - Electronic Theses Online ServiceGBUnited Kingdo