Abstract
AIM: 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). CONCLUSION: 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.