Using RNA interference to identify the different roles of SMAD2 and SMAD3 in NIH/3T3 fibroblast cells |
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Authors: | Zheng Rong Xiong Qi Zuo Bo Jiang SiWen Li FengE Lei MingGang Deng ChangYan Xiong YuanZhu |
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Affiliation: | Key Laboratory of Swine Breeding and Genetics, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, P. R. China. |
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Abstract: | Smad proteins are principal intracellular signaling mediators of transforming growth factor beta (TGF-beta) that regulate a wide range of biological processes. However, the identities of Smad partners mediating TGF-beta signaling are not fully understood. We firstly examined the expression of Smad2 and Smad3 induced by TGF-beta 1 in normal NIH/3T3 cells. The expression of Smad2 and Smad3 was assessed by RT-PCR and Western blotting. The results showed that the expression of Smad2 was increased after treatment with TGF-betaI, but Smad3 was more sensitive to TGF-betaI than Smad2. RNA interference (RNAi) provides a new approach for elucidation of gene function. Use of hairpin siRNA expression vectors for RNAi has provided a rapid and versatile method for assessing gene function in mammalian cells. Here, we have constructed Smad2 and Smad3 hairpin siRNA expression plasmids, and then transfected them into mouse NIH/3T3 cells. Endogenous Smad2 and Smad3 proteins decreased significantly at 48 h after transfection. We found the expression of Smad3 in Smad2-depleted cells was increased, however, the expression of Smad2 in Smad3-depleted cells was not changed. Consistently, the expression of Smad4 mRNA was also attenuated in Smad3-depleted cells. From these data, we suggest that Smad3, but not Smad2, may play a key role in TGF-beta signaling. |
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Keywords: | RNAi Smad2 Smad3 transforming growth factor‐β (TGF‐β) signal transduction |
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