Background. Endothelial cells growing in high glucose concentration show reduced angiogenic potential, migration and proliferation rate, with a pronounced increase in apoptosis, compared to normal glucose. High glucose is also able to alter the expression of microRNAs, a class of endogenous 22-25nt single-stranded RNA molecules that regulate gene expression post-transcriptionally. Aim. The aim of this thesis is to investigate whether the cellular responses induced by high glucose are mediated by microRNAs and to gain information about the targets and thus the patwhways regulated by glucose-sensitive microRNAs, particularly focusing on miR-492. Methods. We used the transfection of miRNAs over-expressed after high glucose (miR- 125a, miR-320, miR-487b, miR-492) to study their effects on HUVEC angiogenic potential (tube formation) cell proliferation and cell migration. We then used pull-out technique to identify miR-492 targets. Results. The glucose-regulated microRNAs reduced, to different extent, tube formation, migration and proliferation. They also regulated each other's expression, and all of them were able to induce mature miR-487b, thus forming a regulatory circuit. Focusing on miR-492, being the strongest in impairing proliferation, migration and tube formation, we demonstrated that its inhibition can reverse all the phenotypes except for cell migration. We found that eNOS, a known regulator of vascular functions, is a key mediator of miR-492 effects on angiogenesis. We then identified several direct targets of miR-492 that could mediate eNOS regulation, including PDPK1 and SP1. Conclusions. This thesis demonstrated that high glucose-sensitive microRNAs reproduce at different extent the anti-angiogenic phenotypes showed by glucose and that miR-492 is a strong anti-angiogenic microRNA, acting principally on the expression of eNOS and on its signalling pathway.
miR-492 is a glucose-responsive microRNA that affects the angiogenic properties of HUVECs by regulating eNOS transcription and the activation of eNOS signalling pathway
Rainaldi Giuseppe
2011
Abstract
Background. Endothelial cells growing in high glucose concentration show reduced angiogenic potential, migration and proliferation rate, with a pronounced increase in apoptosis, compared to normal glucose. High glucose is also able to alter the expression of microRNAs, a class of endogenous 22-25nt single-stranded RNA molecules that regulate gene expression post-transcriptionally. Aim. The aim of this thesis is to investigate whether the cellular responses induced by high glucose are mediated by microRNAs and to gain information about the targets and thus the patwhways regulated by glucose-sensitive microRNAs, particularly focusing on miR-492. Methods. We used the transfection of miRNAs over-expressed after high glucose (miR- 125a, miR-320, miR-487b, miR-492) to study their effects on HUVEC angiogenic potential (tube formation) cell proliferation and cell migration. We then used pull-out technique to identify miR-492 targets. Results. The glucose-regulated microRNAs reduced, to different extent, tube formation, migration and proliferation. They also regulated each other's expression, and all of them were able to induce mature miR-487b, thus forming a regulatory circuit. Focusing on miR-492, being the strongest in impairing proliferation, migration and tube formation, we demonstrated that its inhibition can reverse all the phenotypes except for cell migration. We found that eNOS, a known regulator of vascular functions, is a key mediator of miR-492 effects on angiogenesis. We then identified several direct targets of miR-492 that could mediate eNOS regulation, including PDPK1 and SP1. Conclusions. This thesis demonstrated that high glucose-sensitive microRNAs reproduce at different extent the anti-angiogenic phenotypes showed by glucose and that miR-492 is a strong anti-angiogenic microRNA, acting principally on the expression of eNOS and on its signalling pathway.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
