Role And Mechanism Of HtrA1 In Blue-light-irradiated Human RPE Cells

Part1 Eastablish of light-injured model and expressin of HtrAl in light-injured human retinal pigment epithelial cellsPurpose:To eastablish the light-injured model and investigate the expressin of HtrAl in light-injured human retinal pigment epithelial cells.Methods:Cultured human retinal pigment epithelial cell ARPE-19 were exposed to the blue light at the intensity of(2000 ± 500)Lux for 6 h to establish the light injured model.Apoptosis of the light injured model was assessed by flow cytometry.Real-time PCR and Western blot were used to detect the changes of HtrAl mRNA and protein expression at 12h,24h,48h and 72h after light exposure.Light injured cells were divided into HtrAl siRNA group,negative control group and blank control group.HtrAl siRNA group and negative control group were transfected with HtrAl siRNA and control siRNA respectively.The siRNA interference efficiency was detected by Real-Time PCR and Western blot.Results:The apoptotic rate of ARPE-19 cells in light injured model was significantly higher than that of normal cells(P<0.05).Real-Time PCR results showed that the mRNA levels of HtrAl in light-injured model was significantly higher than that in normal cells at different time points(P<0.05).Western blot results showed that the protein levels of HtrAl in the light injured model increased significantly compared to that in normal ARPE-19 cells at 24h,48h and 72h after light exposure(P<0.05).The expression of HtrAl mRNA and protein in normal cells did not change at different time points.Compared with negative control group and blank control group,the expressin of HtrAl in HtrAl siRNA group was decreased significantly(P<0.05).Conclusion:Administration of blue light can result in damage of ARPE-19 cells,which is similar to age-related macular degeneration.Blue light can gradually increase the expression of HtrAl.HtrAl RNAi can effectively silence HtrAl gene expression.Part2 Inhibition of cell proliferation,migration and apoptosis in blue-light-irradiated human RPE cells by down-regulation of HtrAlPurpose:To investigate the effect of HtrAl on the proliferation,migration and apoptosis of human retinal pigment epithelium line ARPE-19 cells in the light injured model,as well as the expression of the apoptosis related molecules.Methods:The human retinal pigment epithelium cell line ARPE-19 was exposed to blue light to establish the light injured model.The cells were transfected with HtrAl siRNA to knockdown HtrAl expression.Subsequent expression of HtrAl was determined by Real-Time PCR and Western blot,respectively.Changes in cell proliferation,migration and apoptosis were assessed by CCK-8,transwell assay and flow cytometry respectively.The changes in the mRNA and protein levels of Bax,Caspase-3 and Bcl-2 expression were detected by Real-Time PCR and Western blot respectively.Results:HtrAl was highly expressed in ARPE-19 cells after blue light irradiation.Compared with control groups,the cell proliferation and migration ability of the HtrAl siRNA group were significantly decreased(P<0.05).The apoptotic rate of the HtrAl siRNA group was significantly lower than that of the negative control group and the blank control group(P<0.05).Cells of HtrAl siRNA group mainly remained in G0/G1 phase,the difference was statistically significant(P<0.05).Compared with control groups,Bax and Caspase-3 expression in HtrAl siRNA group were significantly reduced both at mRNA and protein levels,while the mRNA and protein expression of Bcl-2 significantly increased in ARPE-19 cells after HtrAl siRNA interference(P<0.05).Conclusion:Silence of HtrAl may inhibit the proliferation,migration and apoptosis of ARPE-19 cells in light injured model.Moreover,HtrAl suppression in blue-light-irradiated ARPE-19 cells may ameliorate cell apoptosis through down-regulation of Bax and Caspase-3,and up-regulation of Bcl-2 expression.Part3 Mechanisms of effect on vascular endothelial growth factor A in light-injured model with HtrAl silencePurpose:To observe the effect of siRNA-mediated silencing of serine protease HtrA1 on vascular endothelial growth factor-A(VEGF-A)in the light-injured model and to explore its possible mechanism.Methods:Cultured human retinal pigment epithelial cell ARPE-19 were exposed to the blue light at the intensity of(2000 ± 500)Lux for 6 h to establish the light injured model.Light injured cells were divided into HtrA1 siRNA group,negative control group and blank control group.HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively.48h after tranfection,expression of HtrA1.VEGF-A and NF-κB were determined by Real-Time PCR and Western blot respectively.Results:The mRNA level of HtrA1,VEGF-A and NF-κB in HtrA1 siRNA group were significantly lower than that in negative control group and blank control group(P<0.05).The protein level of HtrA1,VEGF-A and NF-κB in HtrA1 siRNA group were significantly lower than that in negative control group and blank control group(P<0.05).Compared with the normal cells,the expression of HtrA1,VEGF-A and NF-κB in negative control group and blank control group increased significantly both at mRNA and protein levels,the difference was statistically significant(P<0.05).Conclusion:The expressions of VEGF-A and NFκB present a similar decreasing trend with the HtrA1 expression in the HtrA1 siRNA group,indicating that there may be some relationship between them.HtrA1,VEGF-A and NF-κB may be involved in the pathophysiology of light damage,and related process of AMD.