Effect of siRNA interfering SLC7a8 on uptaking L-dopa in NRK-52E cells / 中国病理生理杂志

ABSTRACT

AIM: To investigate the effect of selective silencing of SLC7a8 on uptaking L-dopa in renal tubular epithelial cells of rat (NRK-52E). METHODS: The three siRNAs targeting SLC7a8 (siRNA-1, siRNA-2, siRNA-3) were designed and synthesized. A siRNA with nonspecific coding sequence (siRNA-con) was used for control. All siRNAs were transfected into NRK-52E cells. The siRNA-con transfected group, blank control group and gene-specific silencing SLC7a8 group were set up. The efficiency of transfection was estimated by flow cytometry. The efficiency of RNA interference was detected and screened by RT-PCR preliminarily, and was followed by Western blotting at protein level. The concentrations of L-dopa uptake into the NRK-52E cells were detected by ultraviolet spectrophotometry at different time points (6, 12, 24, 36, 48, 60, 72 and 120 min). RESULTS: The transfection efficiency was 94% detected by flow cytometry. The initial screening of RT-PCR showed that the efficiencies of RNA interference of siRNA-1 and siRNA-3 were higher, and siRNA-3 was the highest at protein level determined by Western blotting. No distinctive change was found between siRNA-con treated NRK-52E and blank control cells. The L-dopa uptake at different time points (6, 12, 24, 36, 48, 60, 72 and 120min) in siRNA-interference group was lower than that in siRNA-con transfected group and blank control group. No significant difference of L-dopa uptake between siRNA-con group and blank control group was observed. CONCLUSION: RNA interference technology selectively down-regulates SLC7a8 expression in rat renal tubular epithelial cells. The L-dopa uptake is also decreased after specifically silencing the slc7a8 expression.