Construction of combined lentiviral vectors for rat Slit2 gene RNA interfering and its down-regulation effects on Slit2 gene in rat retinal pigment epithelial cells

Background

Age-related macular degeneration (AMD) is the leading cause of blindness in people over 50 years old, of which 90% cases are caused by choroidal neovascularization (CNV). Current treatments on AMD have gained great achievements, but there are still some drawbacks.So we need to search for new targets to cure CNV.

Objective

This study was to construct two combined lentiviral vectors for rat Slit2 gene RNA interference (RNAi) and verify its interfering effects on Slit2 gene in rat retinal pigment epithelial (RPE) cells.

Methods

Two specific siRNA sequences targeting towards rat Slit2 gene were designed and were annealed to DNA sequences.The DNA sequences and GV248-enhanced green flourescent protein (EGFP) vectors were combined together as recombinant vectors and then were identified.The GV248-EGFP vector, helper 1.0 and helper 2.0 were transfected together into 293T cells and the two combined lentiviral vectors for rat Slit2 RNAi were gained from the cell supernatant after 72 hours of transfection.The titers of the combined lentiviral vectors were measured.The cells were divided into blank control group, Lv-EGFP vector group, Lv-rSlit2-siRNA1 group and Lv-rSlit2-siRNA2 group.The interference efficacy of the combined lentiviral vectors targeting to rat Slit2 gene were identified by real-time fluorescence quantitative PCR and Western blot.The sequence with higher interference efficacy was transfected to rat RPE cells again.The transfected and nontransfected rat RPE cells were treated with 0, 100, 200 and 400 μmol/L CoCl₂ for the preparation of hypoxia models.The expression of vascular endothelial growth factor-A (VEGFA) mRNA in rat RPE cells was finally measured by real-time fluorescence quantitative PCR and the concentration of VEGFA protein in cell supernatant was assayed by ELISA.

Results

The recombined lentiviral vectors for rat Slit2 gene RNAi were successfully constructed.The titers of the two recombinant sequences were 5×10⁸ TU/ml and 3×10⁸ TU/ml, with the transfected rate ≥70%.The relative expression levels of Slit2 mRNA in the Lv-rSlit2-siRNA1 group and Lv-rSlit2-siRNA2 group were 0.67±0.09 and 0.23±0.11, respectively, which were lower than 1.03±0.31 and 0.92±0.07 in the blank control group and Lv-EGFP vector group (all at P<0.01). The expression levels of Slit2 protein in the Lv-rSlit2-siRNA1 group and Lv-rSlit2-siRNA2 group were 0.62±0.07 and 0.49±0.02, respectively, which were lower than 1.00±0.10 in blank control group and 0.95±0.11 in Lv-EGFP vector group (all at P<0.01). Significant differences were found in the expression of VEGFA mRNA and protein in RPE cells among different concentrations of CoCl₂ groups (mRNA: F_{concentration}=127.998, P<0.01; F_group=69.663, P<0.01.Protein: F_{concentration}=17.059, P<0.01; F_group=91.791, P<0.01), and the expression of VEGFA mRNA and the concentration of VEGFA protein were evidently lower in the Lv-rSlit2-siRNA2 group than those in the blank control group after being treated by 100, 200 and 400 μmol/L CoCl₂ (all at P<0.01).

Conclusions

Recombined lentiviral vector for rat Slit2 gene RNAi is successfully constructed, which can effectively knockdown rat Slit2 gene and inhibit the expression of VEGFA in rat RPE cells.