| We aimed to screen the effective small interference RNAs(siRNAs), construct expression vectors of ubiquitination ligase Arkadia and ubiquitin COOH-terminal hydmlase-L5(also known as UCH37), and investigate the effect of Smad7on key factors Smad2, Smad3and TβRI expressions of TGF-β1/Smads signal pathway in aristolochic acid nephropathy(AAN).After transferring series of different concentrations ratios mixtures of LipofectaminTM2000and control siRNAs into renal tubular epithelial cells(RTECs) of mice, we optimized the transfection condition by observing expressions of green fluorescences and undertaking Real-Time PCR reactions. And we detected mRNA inhibition ratios of Arkadia and UCH37by Real-Time PCR after siRNA transfections. Then we constructed expression vectors pGPU6/GFP/Neo of Arkadia and UCH37, cut enzymes and tested subsequences. The best transfection ratio and time of plasmid DNA and LipofectaminTM2000were screened by Real-Time PCR and CCK-8after transferring Arkadia vector. Then the expression vectors of Arkadia and UCH37were transferred into RTECs of AAN model, and the expressions changes of CK18,vimentin, a-SMA and Smad7were detected by Real-Time PCR and Western blot.The results of fluorescence expressions and Real-Time PCR showed that30pmol siRNA:1.5μL LipofectamineTM2000was the best transfection condition. And the Real-Time PCR results indicated that interferences of Arkadia-1490and UCH37-721were the most obvious. By identification, we found that the inserted sequences of vectors were completely correct. The Real-Time PCR and CCK-8results showed that transfection efficiency of vector displayed dose and time dependents. When the ratio of plasmid DNA:LipofectamineTM2000was0.66μg:2μL, and the transfection time was24h, Arkadia mRNA expression inhibition was very obvious and the cytotoxicity was the lowest. Cells transfected with the pGPU6/GFP/Neo-Arkadia vector showed reduced mRNA and protein levels of vimentin, α-SMA, Smad2, Smad3and TβRI after AAI treatment, while those of CK18and Smad7increased compared with those of untransfected RTECs. Conversely, cells transfected with the pGPU6/GFP/Neo-UCH37vector showed the opposite effect on analyzed signaling molecules after AAI treatment.Arkadia-1490and UCH37-721were the best interference fragments of Arkadia and UCH37, and the pGPU6/GFP/Neo-Arkadia and pGPU6/GFP/Neo-UCH37vectors were successfully constructed. We also found that Smad7decreased expressions of Smad2and Smad3and increased TβRI degradation in AAN. |
