Influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium cells / 中华眼底病杂志

ABSTRACT

Objective To observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells.Methods Cultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000 ± 500) Lux for 6 hours 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.The proliferation of cells was assayed by CCK-8 method.Transwell test was used to detect the invasion ability of these three groups.Flow cytometry was used to detect the cell cycle and apoptosis.The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively.Results The mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62,15.09;P＜0.05).Compared with negative control group and blank control group,the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37,4.52),migration (t =9.56,12.13),apoptosis (t =23.37,29.08) and decreased mRNA (t=17.36,11.32,7.29,4.05) and protein levels (t=12.02,15.28,4.98,6.24) of HtrA1 and VEGF-A.Cells of HtrA1 siRNA group mainly remained in G0/G1 phase,the difference was statistically significant (t=6.24,4.93;P ＜0.05).Conclusion Knockdown of HtrA1 gene may reduce the proliferation,migration capability and apoptosis of light-injured RPE cells,and decrease the expression of VEGF-A.