ABSTRACT
Background Studies showed that microRNA (miR)-375 suppresses the growth,apoptosis,migration and adhesion of tumor cells,and it plays a regulation to the changes of vascular endothelial growth factor (VEGF) in tumor tissue to arrest neovascularization.However,whether miR-375 intervenes the formation of new blood vessel in eyes is unelucidated.Objective This study was to explore the effects of miR-375 on human retinal capillary endothelial cells (HRCECs) function induced by hypoxia.Methods HRCECs were cultured using IMDM and divided into normal control group,CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2 +miR-375 mimic control group,CoCl2+miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group,and hypoxia cell models were created by adding 200 μmol/L CoCl2.MiR-375 and frizzled 4 (FZD4) small interfering RNA (siRNA) were transfected into the cells by 50 nmol/L miRNA lipidosome for 48 hours.The proliferation of the cells was detected by MTT assay;migrated number of the cells was examined by Transwell chamber assay;ELISA was employed to detect the concentrations of VEGF and VE-cadherin in the medium;the expression of β-catenin,cyclinD1,matrix metalloproteinase-2 (MMP2) and VEGF proteins were analyzed by Western blot;tube length of vessel formation was evaluated by Matrigel assay.Cultured cells were divided into normal control group,CoCl2 model group,CoCl2 +mock group and CoCl2 + FZD4 siRNA group,the relative expression of FZD4,a miR-375 targeted gene,was detected by luciferase reporter.Results The relative expression of miR-375 mRNA was significantly increased in the CoCl2 +miR-375 mimic group compared with the CoCl2 + miR-375 mimic control group and reduced in the CoCo2 + miR-375 inhibitor group compared with the CoCo2 + miR-375 inhibitor control group (t =-19.237,8.764,both at P＜0.01),with a higher transfected efficacy for miR-375.The cell proliferative fold,migrated cell number,VEGF and VE-Cadherin contents in the medium and the tube length were significantly different among the CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2 +miR-375 mimic control group,CoCl2 +miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group (F =24.324,26.776,14.113,19.225,15.040,all at P＜0.001),and those in the CoCl2 +miR-375 mimic group were evidently reduced in the CoCl2 +miR-375 mimic group compared with the CoCl2 +miR-375 mimic control group,while those in the CoCl2 +miR-375 inhibitor group were considerably elevated in comparison with the CoCl2 +miR-375 inhibitor control group (all at P＜0.01).The expressions of β3-catenin,cyclinD1,MMP2 and VEGF protein were significantly different among the normal control group,CoCl2 model group,CoCl2 +miR-375 mimic group,CoCl2+miR-375 mimic control group,CoCl2 +miR-375 inhibitor group and CoCl2 +miR-375 inhibitor control group (F=11.753,13.283,16.770,10.334,all at P＜0.001).In addition,the cell proliferative fold,migrated cell number and the tube length were significantly increased in the CoCl2 model group and CoCl2+mock group,and those in the CoCl2+FZD4 siRNA group were decreased in comparison with the CoCl2 +mock group (all at P＜0.05).Conclusions MiR-375 inhibits the growth,migration and tube formation ability of HRCECs in hypoxic status probably by regulating the activation of Wnt pathway via directly targeting FZD4.
ABSTRACT
Background Choroidal neovascularization (CNV) is the primary pathogenic cause of many fundus diseases.Oxidative stress injury of retinal pigment epithelial (RPE) cells plays important role in angiogenesis of choroid new blood vessels.Oxidative stress injury can active p75NTR receptor, a member of tumor necrosis factors family,resulting in the proliferation of vascular endothelial cells.However, the mechanisms of vascular endothelial cell proliferation remain unclear.Objective This study was conducted to investigate the effect of p75NTR overexpression on CNV and the relative mechanism.Methods The ARPE-19 cell line was used in this study.RPE cells were transfected with p75NTR receptor overexpressed plasmid, and untransfected cells served as the control group.The transfected results were verified by reverse transcription-PCR and Western blot assay.Viability of the cells over time was determined in the p75NTR receptor plasmid transfected group by using BrdU assay.The percentage of apoptotic cells was detected by flow cytometry using Annexin V-FITC/PI fluorescence staining.The percentage of reactive oxygen species (ROS) expression in the cells was detected by using H2 DCFDA fluorescence and flow cytometry.Mitochondrial membrane potential and cytochrome C expression were examined under the confocal microscope.The protein expressions of cleaved caspase-3, Fas and VEGF were determined by Western blot assay.Results The relative expression level of p75 NTR receptor mRNA was (6.11 ±0.77) times higher than that of the control group, and relative expression level of p75NTR receptor protein in the cells in the p75NTR receptor plasmid transfected group was (7.42±0.48) times higher than that in the control group (t=11.49 and 23.17 ,both at P＜0.01).The absorbency values of the p75NTR receptor plasmid transfected group were (93.12±0.56) ％ , (86.30±0.66) ％ , (72.53-±0.86) ％ and (60.77 ±2.81) ％ in 12,24,36 and 48 hours after plasmid transfection, which were significantly lower than 100％ in the control group, and the apoptotic percentages were evidently higher than that in the control group (all at P＜0.05).The relative fluorescence intensity of ROS fluorescence in the p75NTR receptor plasmid transfected group was 2.4 times higher than that in the control group,showing significant difference (t=16.45, P＜0.01).The positive expressing rate of mitomarker (mitochondrial membrane potentials) was 100％ in the control group and (37.30± 2.06)％ in the p75NTR receptor plasmid transfected group, with significant difference between them (t =57.71,P＜0.01).The fluorescence intensity of cytochrome C expression was elevated in the p75NTR receptor plasmid transfected group compared with the control group.Compared with the control group,the expressing levels of cleaved caspase-3 ,Fas and VEGF165 proteins in the cells were significantly raised in the p75NTR receptor plasmid transfected group (all at P＜0.01).Conclusions Overexpression of p75NTR receptors in RPE cells leads to mitochondrial damage and cellular apoptosis and the secretion of VEGF protein, which sequentially promote CNV.P75NTR receptor may be another important regulation pathway in RPE oxygen damage.