MiRNA-520c-3p Regulation Of Cell Function And Mechanism In Human Vascular Endothelium

Objective:Atherosclerosis(AS),the most common and harmful disease,is a chronic inflammatory disease occurring in the middle and large arteries.Meanwhile,the apoptosis of endothelial cells lead to the necrosis of atherosclerotic plaque cap,which causes a cap exfoliation thrombosis.Therefore,the precaution of apoptosis of endothelial cell is the key to prevent AS.MiRNA-520c-3p,a newly discovered single-stranded small molecule RNA in tumors,belongs to the family of miRNA-520/373.As a regulator,miRNA-520c-3p regulates the expression of many post-transcriptional genes.Myosin light chain kinase(MLCK)regulates the function of endothelial cells.It induces the contraction of actin by phosphorylating myosin light chain(MLC),weakens endothelial cell-cell adhesion and maintains the balance of vascular endothelial barrier function.Djanybek has been reported that miRNA-520c-3p can inhibit the expression of MLCK in pulmonary endothelium.Previous studies showed that MLCK could regulate the proliferation and migration of vascular smooth muscle cell by related miRNAs and MLCK inhibitors could significantly inhibit the progression of atherosclerosis.Further,our preliminary experimental results showed that the expression of miRNA-520c-3p was significantly down-regulated in PDGF-induced HUVECs.In this study,we mainly studied the endothelial cell functions of miRNA-520c-3p in vascular endothelial cells,meanwhile,to explore the molecular mechanism of vascular endothelial cells in the development of atherosclerosis,which would provide the new idea and theoretical foundation for the inflammation disease such as the atherosclerosis of this study.Methods:(1)The difference expression level of miRNA-520c-3p were measured by Real-time Quantitative PCR(q-PCR)in human umbilical vein endothelial cells(HUVECs)after treatment with platelet-derived growth factor(PDGF);(2)Liposome transfection was used to transfect miRNA-520c-3p mimics up-regulation and inhibitor down-regulation respectively in HUVECs cells,and we analyzed the expression of miRNA-520c-3p by q-PCR measure;(3)It took CCK8 assay used to detect the effect of the miRNA-520c-3p up-regulating or down-regulating on cell proliferation in HUVECs;(4)Cell adhesion assay was used to detect the effect of up-regulating or down-regulating of miRNA-520c-3p on the function of adhesion in HUVECs;(5)qPCR and western blot were used to study the effects regulation both in mRNA and protein level in target protein RELA,adhesion molecule ICAM-1 and VCAM-1,proliferation-related proteins and apoptosis-related proteins;(6)Flow cytometry was used to evaluate the effect of the miRNA-520c-3p up-regulation and down-regulation on endothelial cell apoptosis.;(7)It took immunofluorescence method to detect the cytoplasmic localization of protein RELA in HUVECs;(8)The effects of miR-520c-3p on AKT/NF-?B signaling pathway was detected by western blot.Results:(1)PDGF induced the adhesion between vascular endothelial cells and monocytes by promoting the expression of inflammatory molecules ICAM-1 and VCAM-1,and it promoted cell apoptosis and inhibited cell proliferation by up-regulating the expression of apoptosis-related proteins BAX,Caspase-3 and Caspase-9;(2)With the stimulation of PDGF,the expression of miRNA-520c-3p was down-regulated and the expression of RELA was up-regulated,and there was the negative correlation between the expression of mRNA and protein level;(3)The cell adhesion results showed that miRNA-520c-3p significantly reduced the adhesion ability between HUVECs and THP-1 by down-regulating the expression of molecules ICAM-1,VCAM-1 and inflammatory factors IL-6 and TNF-?;(4)miRNA-520c-3p promoted the proliferation and repair and inhibited the apoptosis of vascular endothelial cells by down-regulating the expression of apoptosis-related proteins;(5)The siRNA interference technology results showed that si-RELA inhibited the adhesion between HUVECs and THP-1 cells and the expression of inflammatory related molecules and inhibited the apoptosis of endothelial cells and modulated the expression of apoptosis-related proteins,and it promoted the proliferation and repair of vascular endothelial cells;(6)si-RELA could restore the effect of adhesion promoting by miRNA-520c-3p inhibitor between the vascular endothelial cells and monocyte macrophages,and restore the up-regulation effect of miRNA-520c-3p inhibitor on apoptosis-related proteins BAX,Caspase-3 and Caspase-9;(7)miR-520c-3p regulates the nuclear distribution of RELA,i.e.inhibits the transfer of RELA into the nucleus;(8)miRNA-520c-3p inhibited the proteins phosphorylation of AKT and RELA,and si-RELA restored the phosphorylation of AKT and RELA effect of proteins promoted by miR-520c-3p inhibitor,and the results suggested that miRNA-520c-3p might regulate the proliferation,apoptosis and adhesion through AKT/NF-?B signaling pathway of vascular endothelial cells.Conclusion:(1)miRNA-520c-3p targeting RELA inhibits the adhesion and apoptosis of vascular endothelial cells and promotes cell proliferation;(2)miRNA-520c-3p targeting RELA through AKT/NF-?B signaling pathway regulates the proliferation,apoptosis and adhesion of vascular endothelial cells.