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TGF-1 RNA Interference in Mouse Primary Dura Cell Culture: Downstream Effects on TGF Receptors, FGF-2, and FGF-R1 mRNA Levels

Gosain, Arun K. M.D.; Machol, Jacques A. IV B.A.; Gliniak, Christy B.S.; Halligan, Nadine L. N. M.S.

Plastic & Reconstructive Surgery:
doi: 10.1097/PRS.0b013e3181b98947
Experimental: Original Articles
Discussion
Abstract

Background: Transforming growth factor (TGF)-β1 and fibroblast growth factor (FGF)-2 have both been shown to have significant roles in the regulation of murine calvarial suture fusion. Methods to decrease gene expression of these cytokines and their respective receptors have been established, but because of side effects, clinical applications are limited. In this study, the authors examined the effect of TGF-β1–specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-β1, its TGF-βR1 and TGF-βR2 receptors, and FGF-2 and its R1 receptor in murine dura cells.

Methods: A primary dura cell line was established from CD-1 mice. Transfection efficiency using Lipofectamine was determined using BLOCKiT. Dura cells were transfected with serial concentrations of TGF-β1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-β1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction.

Results: Optimal inhibition of TGF-β1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-β1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-βR1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-βR2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4.

Conclusions: These results indicate that TGF-β1 siRNA has the potential to alter the murine dura cytokines responsible for suture fusion in vitro. Manipulating underlying cranial suture biology with siRNA technology may ultimately allow control over suture fusion. This intervention may ultimately function as an effective adjunct to surgical intervention for craniosynostosis.

Author Information

Cleveland, Ohio; and Milwaukee, Wis.

From the Department of Plastic Surgery, Case Western Reserve University School of Medicine, and the Department of Plastic Surgery, Medical College of Wisconsin.

Received for publication September 22, 2008; accepted May 20, 2009.

Disclosure: None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this article.

Arun K. Gosain, M.D., Department of Plastic Surgery, Case School of Medicine, University Hospital (Lakeside), MS 5044, 11100 Euclid Avenue, Cleveland, Ohio 44106, arun.gosain@uhhospitals.org

©2009American Society of Plastic Surgeons