The Effect Of MiR-320 On Low Shear Stress Induced EndMT And Its Potential Mechanisms

Pathological changes of Atherosclerosis(Atherosclerosis,As)starting from the endometrium,often occurs in blood vessels,bending,area of the bifurcation As low shear stress or shock shear stress area.Low shear stress-induced endothelial mesenchymal transition plays an important role in As,but its regulatory mechanism is not yet clear.When endothelial cells undergo endothelial cell transformation,the tight junctions between endothelial cells are destroyed,and cell proliferation,invasion,and migration capabilities increase.Studies have found that overexpression of miR-320 can inhibit endometrial cancer cell proliferation,cell migration,cell cycle and invasion.Therefore,this article will explore the relationship between miR-320 and low shear stress-induced endothelial mesenchymal transformation and its regulatory mechanism.[Objective]: To investigate the role of miR-320 in low shear stress-induced endothelial-mesenchymal transition and its regulatorymechanism.[Methods]: By transfecting miR-320 mimics or miR-320 inhibitors,vascular endothelial cells that overexpress miR-320 vascular endothelial cells and miR-320 expression were constructed.Under static culture conditions,the experimental groups were: blank control group,miR-320 overexpression group(miR-320 mimics),miR-320 overexpression control group(mimics control);blank control group,miR-320 inhibition group(miR-320 inhibitor),miR-320 inhibitor control(inhibitor control),RT-qPCR detection of miR-320 levels.Western bott Western blot detection of vascular endothelial cell surface antigen CD31,VE-Cadherin expression,mesenchymal cell surface antigen ?-SMA,Vimentin expression,SOX4,Wnt / ?-Catenin signaling pathway downstream target genes ?-Catenin,CD44 Expression of Cyclin D1,AXIN2.The scratch experiment and transwell experiment detected the effect of miR-320 on the migration and invasion of vascular endothelial cells.The effect of rhodamine phalloidin staining on the vascular endothelial cytoskeleton.CCK8 and MTT experiments were used to detect the effect of miR-320 on the proliferation of vascular endothelial cells,and flow cytometry was used to detect the vascular endothelial cell cycle.The parallel plate flow cavity system was used to perfuse for 6 hours under low shear stress(4dyn / cm2).The experimental groups were as follows: blank control group,miR-320 overexpression group(miR-320 mimics),miR-320 overexpression control group(mimics control);blank control group,miR-320 inhibitor group(miR-320 inhibitor),miR-320 inhibitor control group(inhibitor control).[Results]: miR-320 mimics-treated endothelial cells miR-320 levels were significantly increased.Under static culture,miR-320 inhibitor down-regulated the expression of endothelial cell markers VE-Cadherin and CD31,and up-regulated the expression of interstitial cell markers Vimentin and ?-SMA.miR-320 mimics up-regulated the expression of VE-Cadherin and CD31,and inhibited the expression of Vimentin and?-SMA,indicating that miR-320 overexpression can inhibit endothelial mesenchymal transformation.miR-320 inhibitor up-regulates the expression of downstream target genes ?-Catenin,Cyclin D1,CD44 of Wnt / ?-Catenin signaling pathway,and inhibits the expression of AXIN2,indicating that miR-320 inhibitor activates Wnt / ?-Catenin signaling pathway.The HUVECs in the miR-320 inhibitor group had thickened cytoskeletal microfilaments and increased stress fibers.The miR-320 mimics group had thinner microfilaments and decreased stress fibers.miR-320 inhibitor promotes the migration of HUVECs,but has no significant effect on the proliferation and proliferation of HUVECs.Bioinformatics prediction analysis indicates that SOX4 may be the target gene regulated by miR-320.Western blot test results confirmed that miR-320 mimics down-regulated SOX4 expression and miR-320 inhibitor up-regulated SOX4 expression.miR-320 inhibitor further inhibits the expression of endothelial cell surface markers VE-Cadherin and CD31 under low shear stress,further upregulates the expression of interstitial cell surface markers Vimentin and ?-SMA under low shear stress,and Wnt / ?-The downstream expression of target genes ?-Catenin,Cyclin D1 and CD44 in Catenin signaling pathway increased,while AXIN2 decreased,indicating that inhibition of miR-320 further enhanced Wnt / ?-Catenin signaling pathway to promote low shear stress-induced endothelial mesenchymal transition.miR-320 mimics up-regulates the expression of endothelial cell surface markers VE-Cadherin and CD31 under low shear stress,inhibits the expression of interstitial cell surface markers Vimentin and ?-SMA under low shear stress,and ?-Catenin and Cyclin D1 The expression of CD44 was decreased,while the increase of AXIN2 indicated that miR-320 overexpression partially reversed the low shear stress-induced endothelial mesenchymal transition by inhibiting the Wnt / ?-Catenin signaling pathway.[Conclusion]: miR-320 may regulates endothelial mesenchymal transition induced by low shear stress through the SOX4 / ?-catenin signaling pathway.