[The influence of down-regulation of focal adhesion kinase by RNA interference on the adhesion and migration of rat hepatic stellate cells in vitro].
OBJECTIVE
To investigate the role of focal adhesion kinase (FAK) in adhesion and migration of hepatic stellate cells (HSC).


METHODS
Two recombinant plasmids expressing short hairpin RNAs (shRNAs) targeting FAK were constructed and one plasmid substantially suppressing FAK expression in HSC was selected. Real-time PCR and Western blot were used to detect the knockdown effects of FAK gene. After 48-hour treatment with FAK shRNA, toluidine blue colorimetric assay was used to detect the cell adhesion. Wound-healing assay and improved Boyden double-chamber were used to detect the cell migration induced by FN.


RESULTS
The recombinant plasmid expressing FAK shRNA was successfully constructed and transfected into HSC. Compared with the controls, the expression of FAK mRNA and protein in HSC treated with FAK shRNA was markedly down-regulated by 76.82% and 72.53%, respectively. The expression of p-FAK (Tyr397) protein was also decreased by 62.71% 48 h posttransfection. The adhesion of HSC was inhibited by 58.69% at 48 h after shRNA transfection. FAK gene silencing could also dramatically inhibit FN-stimulated HSC migration, and the cell migration distance and the cell number of crossing membrane were decreased by 58.27% and 83.70%, respectively.


CONCLUSIONS
FAK gene silencing suppresses adhesion and migration of HSC, and FAK may be a potential target for novel anti-fibrosis therapies.
An
Jun-yan
JY
Department of Gastroenterology, the Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang 050000, China.
Zhang
Xiao-lan
XL
Yao
Dong-mei
DM
Dun
Zhi-na
ZN
Xie
Shu-rui
SR
Hao
Li-sen
LS
D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement; D015536:Down-Regulation; D005353:Fibronectins; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000201:enzymology*; D008103:Liver Cirrhosis / Q000473:pathology / Q000517:prevention & control*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014162:Transfection
D005353:Fibronectins; D012333:RNA, Messenger; D051417:Focal Adhesion Kinase 1; C496052:Ptk2 protein, rat