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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(QPCR) and Western blotting analysis.The production of type collagen and type 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 collagen and type collagen mRNA transcripts in FAK shRNA plas-mid group were signif icantly 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 signif icantly 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-f ibrosis therapies.
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 (QPCR) and Western blotting analysis. The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR. 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. plasmids into HSC resulted in disrupted FAK expression. Compared with the HK group, the levels of type collagen and type collagen mRNA transcripts in FAK shRNA plas-mid group were signif ically 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 signif icantly 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) .Wowever, 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-f ibrosis therapies.