Regulatory Role Of MicroRNA-181b In The Angiogenesis Of Senescent Endothelial Cells

Background and AimsAngiogenesis is the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis in tissues exposed to severe ischemia is a major survival mechanism against hypoxic tissue injury. Aging is associated with various changes in the vascular system at differing structural and functional levels, which may not only increase the risk of atherosclerosis but also affect the prognosis of ischemia diseases. Senescent endothelial cells cease to proliferate and undergo other functional changes in association with aging. Reduced proliferation and migration may limit the capacity to form new vascular structures.MicroRNAs (MiRNAs) play a key role in angiogenesis and endothelial senescence. It is reported that miR-181b is upregulated in senescent endothelial cells. But the role of miR-181b in senescent endothelial cells and the mechanism is still unclear. Silent information regulator1(SIRT1) is predicted to be a target of microRNA-181b. SIRT1is downregulated in senescent endothelial cells and protects against endothelial dysfunction. And it is regulated by many microRNAs. The present study was designed to study the role of miR-181b in angiogenesis of senescent endothelial cells and its mechanism.Methods1. Primary human umbilical vein endothelial cells (HUVECs) were cultured and population-doubling levels (PDLs) were calculated during passages. PDL44was identified as senescent HUVECs.2. To compare the expression of miR-181b in PDL8and PDL44HUVECs, miR-181b was dected by real-time PCR. To observe the effect of miR-181b in PDL44HUVECs, miR-181b mimics and inhibitor were separately transfected into HUVECs (PDL44), and then the abilities of cell proliferation, migration and tube formation were assayed by MTS test, scraching test, and tube formation test, respectively.3. To investigate the mechanism of miR-181b in regulating endothial cell proliferation and migration, the expression of SIRT1was detected by real-time PCR, western blot and luciferase reporter assay.Results1. Compared with PDL8HUVECs, the percentage of SA-β3-gal-positive cells was increased in PDL44HUVECs by4.7-folds (P＜0.001). The expression of p53and p21was upregulated in PDL44HUVECs by69%(P=0.003) and61%(P=0.01), respectively. And the telomere length was shorter in PDL44HUVECs by42%(P=0.01).2. Compared with PDL8HUVECs, the expression of miR-181b was increased in PDL44HUVECs by64%(P=0.04). In vitro analysis of PDL44HUVECs, overexpression of miR-181b inhibited endothelial proliferation by18%(P<0.001) and migration by40%(P=0.03), while inhibitions of miR-18b can significantly increase endothelial proliferation and migration. The regulatory role of miR-181b in endothelial tube formation was not observed in response to VEGF stimulus.3. Neither overexpression nor inhibition of miR-181b in PDL44HUVECs affected the expression of SIRT1. Also, results from dual-luciferase assay indicated that miR-181b did not target the3’-UTR of SIRT1.ConclusionInhibition of miR-181b can enhance proliferation and migration of senescent endothelial cells, which it may serve as a target in regulating senescent endothelial repair and angiogenesis. Our results indicate that miR-181b may regulate proliferation and migration of senescent endothelial cells by other targets, but not SIRT1.