Effect of Numb gene on tubular epithelial-to-mesenchymal transition in rat proximal epithelial cells / 中华肾脏病杂志

ABSTRACT

Objective To explore the effect of Numb on tubular epithelial-to-mesenchymal transition (EMT) in rat proximal epithelial cells. Methods NRK52E cells were treated with different concentrations of recombinant human transforming growth factor-β1 (TGF-β1) (0, 1, 5, 10, 15, 20 μg/L) for 48 h or 10 μg/L TGF-β1 for different times (0, 24, 48, 72 h) in vitro. The expressions of E-cadherin, a-smooth muscle actin(α-SMA) and Numb in NRK 52E cells were detected by RT-PCR, Western blot and immunofluorescence staining. Meanwhile Numb siRNA oligo was transfected into NRK 52E cells with lipofectamine before TGF-β1 treatment, then Western blot was applied to detect the protein expression of E-cadherin, α-SMA and Numb in NRK52E cells. Results TGF-β1 could induce EMT in NRK52E cells in dose- and time-dependent manner. During the progress of TGF-β1-induced EMT, the protein expression of Numb in 5, 10, 15, 20 μg/L group was 1.33 folds (P=0.024), 1.39 folds (P=0.035), 1.45 folds (P=0.025), 1.51 folds (P=0.000) respectively as compared to 0 μg/L group. Likewise, the protein and mRNA expression of Numb in 24 h, 48 h, 72 h group was 1.48 folds (P=0.046) and 1.56 folds (P=0.012), 1.54 folds (P=0.011) and 1.82 folds (P=0.008), 1.79 folds (P=0.028) and 1.82 folds (P=0.002) respectively as compared to 0 h group. Moreover, large amount of Numb was accumulated in the cytoplasm. Down-regulation of Numb expression by siRNA transfection did not influence the basal expression of E-cadherin and α-SMA in NRK 52E cells, but attenuated the progression of EMT in NRK52E cells induced by TGF-β1. The up-regulation of α-SMA protein was reduced to 18.1% (P=0.004) while the down-regulation of E-cadherin protein was reversed to 2.19 folds (P=0.004). Conclusion Numb can promote EMT in rat proximal epithelial cells.