Reciprocal Antagonism Between MiR-138 And SIRT1 And Its Implications For The Angiogenesis Of Endothelial Cells

Objective:To understand the change mode of cellular division ability and expression pattern of microRNA-138 during endothelial cells’long term incubation, we set up endothelial cells senescent model and further investigated the role microRNA-138 during this process.Methods:The HUVECs were sub-cultured at a ratio of 1:3 once per week for up to 4 weeks and designated each week’s cells as P1 P2, P3, and P4. We tested the proliferation rate of every week’cells at indicated time points (0,24,48, and 72 h) by CFSE FACS. We tested the expression pattern of microRNA-138 during this process by qRT-PCR. The we divided the cells between two groups:experimental groups transfected with microRNA-138 mimics and control groups transfected with microRNA-control mimics. We examined the effects of the modulation of microRNA-138 levels on the activity of SA-β-Gal of endothelial cells, which is a biomarker for cell senescence. We examined the difference of proliferation rate between two groups. Finally, we performed the cell cycle test to determine whether the effect of miR-138 on endothelial cell was mediated by cell cycle arrest.Results:During the passage from P1to P4, the cellular division ability of HUVECs progressively declined, at the same time, the results of microRNA-specific qRT-PCR showed that the expression level of endogenous miR-138 increased during long-term incubation, with the lowest expression level at P1 and the highest at P4. Overexpression of miR-138 obviously promoted the senescence in HUVECs. The up-regulation of miR-138 restrained endothelial cell proliferation obviously. The overexpression of miR-138 in HUVECs diminished the percentage of cells in the G2/M phase from 29%to 11.84%and increased the percentage of cells in the G1/GO phase from 55.99%to 75.83%Conclusion:During long term incubation, endothelial cells entered the state of senescence and were deprived of the ability of proliferation progressively. The expression level of endogenous miR-138 increased during long-term incubation. Overexpression of miR-138 could accelerate the level of cellular senescence in HUVECs and inhibited the proliferation of endothelial cells by cell cycle arrest.Objective:Previous study indicated that microRNA-138 could inhibit the proliferation rate of endothelal cells. In this part, we investigated the role of microRNA-138 in the regulation of angiogenesis through in vitro and in vivo vascular formation experiments.Methods:We performed endothelial cell migration, tube formation and three-dimensional spheroidal assay to investigate the role of microRNA-138 in endothelial angiogenesis in vitro and Matrigle plug assays and retinal angiogenesis assays to investigate the role of microRNA-138 in endothelial angiogenesis in vivoResults:We found that overexpression of microRNA-138 could reduce the migration ability of endothelial cells, diminished the tube-like structures on the surface of matrigel and lessened the sprouting activity of HUVEC spheroids in collagen gels. In matrigel plug assay, we found that microRNA-138 can counteract the angiogenesis in the matrigel plug induced by VEGF. In the retinal angiogenesis assay, we found that microRNA-138 could inhibit postnatal retinal neovascularization.Conclusion:These results indicate that microRNA-138 can suppress angiogenesis of endothelial cells in vitro and in vivo.Objective:Previous study demonstrated that microRNA-138 could regulate angiogenesis of endothelial cells, but it remained unclear that how this happen. We discussed the underlying mechanism of this phenomenon about how microRNA-138 regulated angiogenesis.Methods:We performed an in silico search to screen for target genes. We performed luciferase reporter assay to confirm whether microRNA-138 could regulate its target gene through 3’UTR of target gene. We performed WB and PCR assay to investigate whether microRNA-138 can regulate its target gene in the endothelial cells. We performed rescue assay to confirm whether the regulation of angiogenesis by microRNA-138 was mediated by downregulating its target gene.Results:We found that SIRT1was the target gene of microRNA-138 and its 3’UTR included a perfect matching region for microRNA-138. Luciferase reporter assay showed that microRNA-138 could regulate SIRT1 through its 3’UTR. In endothelial cells, we demonstrated that microRNA-138 can reduce the mRNA and protein level of SIRT1. Overexpression of SIRT1 could rescue miR-138 induced decrease in cell proliferation and tube formationConclusion:MiR-138 regulated endothelial function through inhibition of SIRT1 expression.