Impact of gene silencing of ubiquitin-ribosomal protein 52 on the transforming growth factor β1/Smad signaling pathway in rat tubular epithelial cells under high glucose condition

Objective To investigate the change of transforming growth factor (TGF) β1/Smad signaling pathway in rat tubular epithelial cell (NRK-52E) under high glucose condition after gene silencing of ubiquitin-ribosomal protein 52 (UBA52). Methods Firstly, NRK-52E cells were stimulated with 20 mmol/L (normal control group) or 30 mmol/L glucose for 48 hours respectively, while cells cultured with 5.6 mmol/L glucose with or without 24.4 mmol/L of mannitol were set as normal control or hyperosmotic control. Then the mRNA expression of UBA52, TGF-β1 and Smad7 were detected by using real time polymerase chain reaction (PCR) in the 4 groups. After that, three UBA52 small interfering RNA (siRNA) sequences were selected. The transfection efficiency was observed by fluorescence microscope after transfection, and the expression of UBA52 mRNA was detected by real time PCR. The most effective siRNA was selected to be used for subsequent experiments. Then cells stimulated with 30 mmol/L glucose were transfected by UBA52 siRNA for 48 h, those without transfection or being transfected with irrelevant RNA were set as control or negative control. Then the mRNA and protein expression of UBA52, TGF-β1 and Smad7 were detected in the 3 groups. Above mentioned indexes were statistically analyzed by randomized blocks analysis of variance. Results In glucose intervention experiment, there were significant differences in the expressions of UBA52, TGF-β1 and Smad mRNA among the 4 groups (normal control group, hypertonic control group, medium concentration glucose and high concentration glucose group) (F=60.914, 65.112, 29.390, P<0.05); expressions of UBA52, TGF-β1 of medium and high concentration glucose group were significantly higher than those in normal control and hypertonic group, but Smad7 were significantly lower the two groups. In transfection experiment, expressions of TGF-β1 mRNA in blank control group, negative control group and transfection group were 1.00±0.04, 0.98±0.04, 0.49±0.14 respectively (F=26.364, P<0.001), it was significantly lower in the transfection group than that in the other two groups; the expressions of Smad7 mRNA in the 3 groups were 1.00±0.12, 1.05±0.17, 2.24±0.31, respectively (F=22.816, P<0.001). The expressions of TGF-β1 protein of the 3 groups were 0.676±0.357, 0.613±0.011, 0.184±0.073, respectively (F=96.433, P<0.01), it was the lowest in transfection group; the expressions of Smad2/3 protein were 0.68±0.10, 0.63±0.05, 0.27±0.15, respectively (F=12.194, P<0.05), it was the lowest in transfection group; the expressions of Smad7 protein were 0.52±0.05, 0.55±0.12, 0.83±0.06 in the 3 groups, respectively (F=12.154, P<0.05), it was the highest in transfection group. Conclusion The intervention of TGF-β1/Smad signaling pathway of renal tubular epithelial cells under high glucose by UBA52 gene silence may be a new target for the intervention of renal interstitial fibrosis in diabetic nephropathy. Key words: Ubiquitin-ribosomal protein 52; Gene silence; Transforming growth factor β1