Effect of Slit2 gene transfection on vascular endothelial growth factor in retinal pigment epithelium cells and its mechanism

Abstract:

Objective：To investigate the effect of Slit2 gene on vascular endothelial growth factor（VEGF）in retinal pigment epithelium（RPE）cells and the function of PI3K/AKT and MEK/ERK signaling pathways during the course. Methods：（1）human APRE-19 cells were cultured in vitro. （2）RPE cells were transfected by adenovirus-mediated Slit2（Ad-Slit2），then cultured for 12，24，48 h. （3）Before being transfected by Ad-Slit2，RPE cells were pretreated with MEK inhibitor PD98059 and PI3K inhibitor LY294002 for 1 h. （4）VEGF mRNA expression levels were detected by real-time PCR and the protein expression levels were detected by ELISA. The protein expression levels of p-ERK，p-AKT were detected by Western blot. Results：After RPE cells being transfected with Ad-Slit2，real-time PCR result showed that there were significant statistically differences in VEGF mRNA expression among different time points（F=12.789，P=0.018） and among different groups（F=16.855，P=0.015）；time points and groups were interrelated；at 24 h and 48 h，the VEGF mRNA expression was higher in Ad-slit2 group than in adenovirus group（P24 h=0.002 ，P48 h=0.018）. ELISA results showed that there were significant statistically differences in VEGF protein expression among different time points（F=24.886，P=0.000） and among different groups（F=28.250，P=0.006）；time points and groups were interrelated；at 48 h，the VEGF protein expression was higher in Ad-Slit2 group than in adenovirus group（P=0.003）. Western blot showed that p-ERK and p-AKT protein expressions increased in RPE cells transfected with Ad-Slit2；PD98059 decreased the activation of p-ERK and LY294002 decreased the activation of p-AKT. real-time PCR results showed that there were statistically significant differences in VEGF mRNA expression among different time points（F=32.202，P=0.000） and among different groups（F=15.062，P=0.001）；time points and groups were interrelated（F=8.372，P=0.006）. ELISA results showed there were statistically significant differences in VEGF protein expression among different time points（F=90.703，P=0.000） and among dif－ferent groups（F=69.636，P=0.000）；time points and groups were interrelated（F=4.968，P=0.005）. Conclusion：The expression of the VEGF in RPE cells transfected with Ad-Slit2 can be increased by MEK/ERK and P13K/Akt signaling pathways.