Study On The Function And Mechanism Of Annexin A2 In Human Retinal Endothelial Cells

ObjectiveTo investigate the function and mechanism of Annexin A2(ANXA2)in human retinal endothelial cells(HRECs)and to find new target for the prevention and treatment of retinal neovascular diseases.Methods64 healthy C57BL/6J mice were randomly divided into control group and experimental group of 32 each.Experimental group used a hyperoxia-induced method to construct a classical oxygen-induced retinopathy(OIR)mouse model.In order to verify the success of OIR mice mode,the retinal neovascularization was observed by the technique of lectin GS-IB4 fluorescence staining,western blotting(Western Blot)and real-time reverse transcription polymerase chain reaction(RT-PCR)was utilized to detect the expression of hypoxia-inducible factor-1?(HIF-1?);Using Western Blot and RT-PCR to detect the expression of ANXA2 in OIR mice retina to verify the role of ANXA2 in retinal neovascular disease in vivo.Packaging lentivirus including interfere ANXA2 and overexpressing ANXA2.Western Blot and RT-PCR were used to detect the expression of ANXA2 in HRECs which were infected by corresponding lentivirus.The effect of ANXA2 on the formation of neovascularization in HRECs was detected by EdU cell proliferation assay,Transwell cell migration assay and matrix tube formation assay.The influence of ANXA2 on the cell cycle of HRECs was detected by flow cytometry,then we utilize Western Blot to detect the expression of series cell cycle related protein.The effect of ANXA2 on molecular level of HRECs was detected by gene chip technique,and RT-PCR was used to verify the expression of relatively stable molecules in gene chips;Western Blot and Nuclear Extraction Kit were used to detect the expression of CYLD,p-I?B?,nuclear NF?B subunit P65,and to explore the effect of ANXA2 on CYLD and NF?B signaling pathway.ResultsIn the OIR mouse model established in this experiment,the retinal neovascularization was disorganized and the vascular delamination was unclear compared with the retina of a normal mouse.Meanwhile,the expression level of HIF-1? gene and protein was significantly increased(mRNA t=25.57,P<0.0001),finally,molding rate reached 81%.At the same time,we found that the expression level of ANXA2 gene and protein in the OIR group was significantly increased(mRNA t=26.58,P<0.0001).Compared with the negative control group,the ANXA2 gene and protein expression levels were significantly reduced after silencing ANXA2(mRNA t=5.248,P=0.0063),and the ANXA2 expression level was increased after overexpression ANXA2(mRNA t=28.95,P<0.0001).After silencing ANXA2,cell proliferation,cell migration,and cell tubular structure were significantly reduced(t=10.23,P=0.0005;t=11.27,P=0.0004;t=15.37,P=0.0001).After overexpression ANXA2,the amount of them was increased(t=3.21,P=0.0326;t=20.19,P<0.0001;t=7.155,P=0.0020).The results of flow cytometry were analyzed by ModFit software.Compared with the negative control group,the number of cells in the G1 phase after silencing ANXA2 was increased,and the number of cells in the S phase was decreased(t=5.01,P=0.0074;t=4.51,P=0.0107).After overexpression ANXA2,the number of cells in the G1 phase decreased,and the number of cells in the S phase increased(t=4.275,P=0.0129;t=4.915,P=0.0080);The expression levels of CyclinD1,CDK4 and CDK6 proteins were reduced after disturbing ANXA2(t=2.835,P=0.0471;t=3.434,P=0.0264;t=7.317,P=0.0019),and increased after overexpression of ANXA2(t=4.147,P=0.0143;t=4.009,P=0.016;t=6.710,P=0.0026).There was no significant changes in the expression of CyclinA1,CyclinB1,CyclinE1,and CDK2.Compared with the negative control group,the expression of CYLD gene and protein increased after silencing ANXA2(t=8.44,P=0.0011;t=9.848,P=0.0006),and the expression of CYLD decreased after overexpression ANXA2(t=12.11,P=0.0003,t=6.054,P=0.003).Since CYLD is a major negative regulator of NF?B signaling,we examined protein expression associated with NF?B signaling.The results showed that compared with the control group,the phosphorylation of I?B? and the expression of p65 protein in the nucleus after ANXA2 interference were reduced(t=7.29,P=0.0019;t=4.901,P=0.008),and the expression of both ANXA2 overexpression was increased(t=3.474,P=0.0255;t=6.069,P=0.0037).ConclusionAnnexin A2 can enhance the function of vascular remodeling in human retinal endothelial cells,including cell proliferation,migration and tube formation.It play a pivotal role in above function may be through regulating G1-to-S phase transition during cell cycle progression,inhibiting the activity of ubiquitination hormone CYLD and activating NF?B signaling pathway.