[Effect of small interfering RNA-mediated Smad3 gene silencing on transforming growth factor-beta1-induced bi-directional effects on skin fibroblast proliferation].
OBJECTIVE
To study the role of Smad3 in transforming growth factor-beta1 (TGF-beta1)-induced bi-directional effects on skin fibroblast proliferation.


METHODS
The Smad3 small interfering (siRNA) plasmid was constructed using a pSUPER vector. The efficiency of cell transfection was detected by fluorescence microscopy, and the inhibitory effect of the plasmid was assessed by real-time quantitative RT-PCR and immunohistochemistry. The effect of the plasmid on the fibroblast proliferation and Smad3 binding activity was analyzed by BRDU ELISA and EMSA, respectively.


RESULTS
The transfection efficiency of the plasmid into the cells was 41.2%. The Smad3 siRNA plasmid produced efficient and specific inhibition of the expression of Smad3, and promoted the cell proliferation in a dose-dependent manner and abrogated the bi-directional effect of TGF-beta1 on the cell proliferation and Smad3 binding activity.


CONCLUSIONS
The siRNA targeting Smad3 gene can inhibit the protein expression and RNA transcription of Smad3, and TGF-beta1 exerts bi-directional regulation on fibroblast proliferation by modulating Smad3 activity.
Li
Ping
P
Molecular Biology Center, State Key Laboratory of Trauma, Burns and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, 400042. ping_ping0074@sina.com
Liu
Ping
P
Chen
Xing-Yun
XY
Zhao
Yan
Y
Ning
Ya-Lei
YL
Yang
Lan
L
Zhou
Yuan-Guo
YG
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D012867:Skin / Q000166:cytology; D051900:Smad3 Protein / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
D034741:RNA, Small Interfering; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D053773:Transforming Growth Factor beta1