Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells.
OBJECTIVE
To investigate the effects and mechanism of disruption of focal adhesion kinase (FAK) expression on collagen metabolism in rat hepatic stellate cells (HSC).


METHODS
The plasmids expressing FAK short hairpin RNA (shRNA) were transfected into HSC-T6 cells, and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction (Q-PCR) and Western blotting analysis. The production of type I collagen and type III collagen in FAK-disrupted cells was analyzed by real-time Q-PCR. The level of collagen metabolism proteins, including matrix metalloproteinases-13 (MMP-13) and tissue inhibitors of metalloproteinases-1 (TIMP-1) was also determined by both real-time Q-PCR and Western blotting analysis.


RESULTS
The transfection of FAK shRNA plasmids into HSC resulted in disrupted FAK expression. Compared with the HK group, the levels of type I collagen and type III collagen mRNA transcripts in FAK shRNA plasmid group were significantly decreased (0.69 +/- 0.03 vs 1.96 +/- 0.15, P = 0.000; 0.59 +/- 0.07 vs 1.62 +/- 0.12, P = 0.020). The production of TIMP-1 in this cell type was also significantly reduced at both mRNA and protein levels (0.49 +/- 0.02 vs 1.72 +/- 0.10, P = 0.005; 0.76 +/- 0.08 vs 2.31 +/- 0.24, P = 0.000). However, the expression of MMP-13 mRNA could be significantly up-regulated by the transfection of FAK shRNA plasmids into HSC (1.74 +/- 0.20 vs 1.09 +/- 0.09, P = 0.000).


CONCLUSIONS
These data support the hypothesis that shRNA-mediated disruption of FAK expression could attenuate extracellular matrix (ECM) synthesis and promote ECM degradation, making FAK a potential target for novel anti-fibrosis therapies.
Dun
Zhi-Na
ZN
Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, No. 215 Heping West Road, Shijiazhuang, Hebei Province, China.
Zhang
Xiao-Lan
XL
An
Jun-Yan
JY
Zheng
Li-Bo
LB
Barrett
Robert
R
Xie
Shu-Rui
SR
D000818:Animals; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics*; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000378:metabolism*; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D024042:Collagen Type I; D024061:Collagen Type III; D034741:RNA, Small Interfering; D019715:Tissue Inhibitor of Metalloproteinase-1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D053509:Matrix Metalloproteinase 13