Inhibition of CCN1 siRNA on retinal endothelial cells

Background

Cysteine-rich 61(Cyr61)/CCN1 has been reported to stimulate retinal neovascularization (RNV) in retinopathy of prematurity (ROP). However, whether CCN1 small interfering RNA (CCN1 siRNA) can inhibit or cure ROP has not been extensively investigated.

Objective

This study was to investigate the regulation effect of CCN1 specific siRNA expression vector on retinal endothelial cells.

Methods

Rhesus choroid-retinal vascular endothelial cells (RF/6A) were cultured under the normoxic (normoxia control group) and hypoxic condition (1%O_2, 5%CO₂ with 94%N₂) in vitro, and then lipofectamine™2000 (LF2000) vector plasmid with or without CCN1 siRNA was transiently transfected in the hypoxic-cultured cells as the CCN1 siRNA transfected group and hypoxic control group, respectively.Reverse transcription PCR was employed to detect the expression of CCN1 siRNA plasmid 24 hours after transfection.The vatality of the cells was assayed by cell counting kit-8(CCK-8) 0, 24, 48, 72 and 96 hours after cultured.Twenty-four hours after cultured, the apoptosis of the cells was evaluated by flow cytometry, and the expressions of CCN1 and vascular endothelial growth factor (VEGF) proteins were detected by immunofluorescence technique and Western blot assay.

Results

The expression band of CCN1 siRNA was detected in the cells 24 hours after transfection of CCN1 siRNA.CCK-8 assay showed that RF/6A cells were significantly increased over time, and the proliferating value (absorbancy) of the cells was significantly reduced in the CCN1 siRNA transfected group compared with in the normoxia control group and hypoxic control group (F_group=198.45, P<0.05; F_time=39.26, P<0.05). The apoptosis rates of the cells were (68.9±1.1)%, (18.9±1.3)% and (39.6±1.8)% in the CCN1 siRNA transfected group, normoxia control group and hypoxic control group, and the apoptosis rates of the CCN1 siRNA transfected group were evidently higher than those of the normoxia control group and hypoxic control group (t=2.93, t=2.56, both at P<0.05). CCN1 and VEGF proteins were weakly expressed in the normoxia control group and strongly expressed in the hypoxic control group, however, their expression intensity was evidently weakened in the CCN1 siRNA transfected group.The related expression levels of CCN1 and VEGF proteins in the CCN1 siRNA transfected group were significantly lower than those in the hypoxic control group (both at P<0.05).

Conclusions

RNA interference targeting CCN1 can inhibit proliferation and promote apoptosis of RF/6A cells.CCN1 siRNA can arrest RNV probably by downregulating the expression levels of CCN1 and VEGF in the cells.

Cysteine-rich proein 61/physiology; Retinal neovascularization/prevention &control; Retinopathy of prematurity/therapy; Anoxia; RNA, small interfering; Vascular endothelial growth factor; Choroid-retinal vascular endothelial cells, Rhesus