Effect of small interfering RNA on the expression of connective tissue growth factor and type I and III collagen in skin fibroblasts of patients with systemic sclerosis.
BACKGROUND
Systemic sclerosis (SSc) is characterized by an excessive production of extracellular matrix. It is widely accepted that fibrosis is induced by transforming growth factor (TGF)-beta in the early stage and is subsequently maintained by connective tissue growth factor (CTGF). CTGF is a cysteine-rich mitogenic peptide that has been involved in various fibrotic disorders and can be induced in fibroblasts by activation with TGF-beta.


OBJECTIVE
To evaluate the effect of small interfering RNA (siRNA) targeting CTGF on the expression of CTGF and type I and type III collagen in SSc.


METHODS
Skin fibroblasts from patients with SSc were cultured in vitro and later transfected using four CTGF-specific siRNAs and one nonspecific siRNA. The effect of CTGF-specific siRNAs on the expression of CTGF and type I and type III collagen was examined and quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry.


RESULTS
Semiquantitative RT-PCR analysis showed that the four CTGF-specific siRNAs significantly reduced CTGF mRNA expression (P < 0.001), of which siRNA742 showed the strongest inhibitory effect with an inhibitory rate of 73%. Three of the four siRNAs could also depress the transcriptional levels of type I and type III collagen mRNA (P < 0.001), of which siRNA742 showed the strongest inhibitory effect with an inhibitory rate of 37% and 29% for type I and type III collagen, respectively. Western blot analysis further demonstrated that three CTGF-specific siRNAs could significantly decrease CTGF protein level (P < 0.001). In addition, immunocytochemical analysis showed that the expression of type I collagen was significantly decreased in fibroblasts after transfection with siRNA742, whereas inhibition of expression of type III collagen was modest.


CONCLUSIONS
Our data for the first time showed that CTGF RNA interference could inhibit expression of CTGF and type I and III collagen in SSc fibroblasts and indicated that CTGF might be an upstream factor regulating type I and type III collagen synthesis, particularly type I collagen. Our findings suggest that silencing CTGF expression might facilitate a potential therapeutic approach for SSc.
Xiao
R
R
Department of Dermatology, Second Xiangya Hospital, Central South University, Changsha, Hunan, China. xiaorong@medmail.com.cn
Liu
F-Y
FY
Luo
J-Y
JY
Yang
X-J
XJ
Wen
H-Q
HQ
Su
Y-W
YW
Yan
K-L
KL
Li
Y-P
YP
Liang
Y-S
YS
D015153:Blotting, Western; D024042:Collagen Type I / Q000378:metabolism*; D024061:Collagen Type III / Q000378:metabolism*; D055513:Connective Tissue Growth Factor; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
C526317:CCN2 protein, human; D024042:Collagen Type I; D024061:Collagen Type III; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor