TGF-beta1 RNA interference in mouse primary dura cell culture: downstream effects on TGF receptors, FGF-2, and FGF-R1 mRNA levels.
BACKGROUND
Transforming growth factor (TGF)-beta1 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-beta1-specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-beta1, its TGF-betaR1 and TGF-betaR2 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-beta1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-beta1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction.


RESULTS
Optimal inhibition of TGF-beta1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-beta1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-betaR1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-betaR2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4.


CONCLUSIONS
These results indicate that TGF-beta1 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.
Gosain
Arun K
AK
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.
Machol
Jacques A
JA
Gliniak
Christy
C
Halligan
Nadine L N
NLN
D000818:Animals; D018929:Cell Culture Techniques; D003393:Cranial Sutures / Q000378:metabolism; D003398:Craniosynostoses / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D004388:Dura Mater / Q000166:cytology / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism* / Q000494:pharmacology; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; D051496:Receptor, Fibroblast Growth Factor, Type 1