The Regulative Effects Of Focal Adhesion Kinase Gene Silencing On Collagen Metabolism In Hepatic Stellate Cells

Liver cirrhosis is one of the most common diseases and frequently-occurring illness which threaten human health. Hepatic fibrosis (HF) is the common pathological changes and end stage of liver cirrhosis. Therefore, how to prevent or reverse HF is the key step to the treatment of various chronic liver diseases and the prevention of liver cirrhosis.At present, the vast majority scholars at home and abroad believed that hepatic stellate cells (HSC) are the major cells responsibling for extracellular matrix (ECM) synthesis, especially collagen synthesis; the central events in the liver fibrogenesis are the activation, proliferation and migration of HSC.Focal adhesion kinase (FAK) is the key signaling molecule in integrin signal pathway. The activated FAK is closely related to the numerous fibrosis diseases, and plays an important role in the occurrence and development of HF. This is consistent with our previous studies, which indicated that the expressions of FAK were increased in the liver tissues of bile duct ligation (BDL) rat HF models and FAK phosphorylation could promote the collagen synthesis of HSC in vivo; Futher, using in vitro cell culture technique, we found that the synthesis of total collagen and collagenⅠin HSC could be inhibited by an endogenous inhibitor of FAK termed FAK related non-kinase (FRNK). So we speculate that FAK gene silencing may represent a novel method and direction for the treatment and reversal of HF.To date, RNA interference (RNAi) is the most effective gene silencing technology, which can specifically inhibit the transcription of target genes, and in turn reduce the expressions and functions of the corresponding proteins.Therefore, in this study, FAK short hairpin RNA (shRNA) plasmids were transfected into HSC transiently in order to further observe the effects of specific inhibition of FAK expression on collagen synthesis of HSC and the mechanisms thereof, hoping that we can deepen the recognition of HF and provide a theoretical basis on the treatment of HF through this experiment.Objective: FAK shRNA plasmids were transiently transfected into fibronectin(FN)-stimulated HSC in vitro, so as to evaluate the effects of specific inhibition of FAK expression on collagenⅠ, collagenⅢsynthesis of HSC, and the dynamic expression of MMP-13,TIMP-1,MT1-MMP,MMP-2 and TIMP-2 in this process.Methods: Using in vitro cell culture technique, FAK shRNA plasmids were transfected into FN-stimulated HSC mediated by cationic polymer, the cell transfection efficiency was analyzed by fluorescent microscope and flow cytometry, and the expressions of FAK in mRNA and protein levels were determined by real-time Q-PCR and Western blot, respectively. The cells were divided into 5 groups: (1) blank control group(Con); (2) FN stimulation group(FN); (3) transfection reagent group(Sofast); (4) pEGFP-HK shRNA group(HK); (5) pEGFP-FAK shRNA group(FAK shRNA). The concentration of FN is 10μg·mL-1 from group (2) to group (5). The levels of CollagenⅠin HSC were assayed by real-time Q-PCR on mRNA level and Western blot on protein level, respectively. Real-time Q-PCR was used to measure the dynamic expressions of CollagenⅢbefore and after FAK shRNA transfection. The expressions of MMP-13, TIMP-1, MMP-2, TIMP-2, MT1-MMP in mRNA and protein levels were determined by real-time Q-PCR and Western blot, respectively.Results: (1) FAK shRNA plasmids were successfully transfected into HSC. Fluorescent microscope and flow cytometry results showed that the transfection efficiency was about 40% at 48 h; Real-time Q-PCR result showed that FAK mRNA was down-regulated by 76.73% at 24 h after FAK shRNA treatment and Western blot analysis showed that the FAK protein was down-regulated by 72.53% at 48 h after FAK shRNA treatment; (2) CollagenⅠmRNA and protein were down-regulated by FAK shRNA: real-time Q-PCR and Western blot results showed that the levels of CollagenⅠin FN group were significantly increased when compared with that of Con group. Compared with the HK group, collagenⅠmRNA can be significantly down-regulated by 64.80% at 36 h after FAK shRNA treatment(0.69±0.03 vs 1.96±0.15), and collagenⅠprotein can be significantly down-regulated by 67.05% at 48 h after FAK shRNA treatment (0.85±0.03 vs 2.58±0.21); (3) CollagenⅢmRNA was down-regulated by FAK shRNA: real-time Q-PCR results showed that collagenⅢmRNA in FN group was obviously increased when compared with that of Con group. Compared with the HK group, the expression of collagenⅢmRNA can be significantly down-regulated by 63.58% at 36 h after FAK shRNA transfection(0.59±0.07 vs 1.62±0.12); (4) MMP-13 mRNA and protein were up-regulated by FAK shRNA: real-time Q-PCR and Western blot results showed that the levels of MMP-13 of FN group were significantly decreased when compared with that of Con group. Compared with the HK group, the expression of MMP-13 mRNA can be significantly up-regulated by 56.96% at 36 h after FAK shRNA plasmids transfected into HSC(1.24±0.04 vs 0.79±0.03), and the expression of MMP-13 protein can be significantly up-regulated by 59.63% at 48 h after FAK shRNA plasmids transfected into HSC(1.74±0.20 vs 1.09±0.09); (5) TIMP-1 mRNA and protein were down-regulated by FAK shRNA: real-time Q-PCR and Western blot results showed that the expression of TIMP-1 of FN group was significantly higher than that of Con group. Compared with the HK group, the expression of TIMP-1 mRNA can be significantly down-regulated by 71.51% at 36 h after FAK shRNA plasmids transfected into HSC (0.49±0.02 vs 1.72 ±0.10), and the expression of TIMP-1 protein can be significantly down-regulated by 67.10% at 48 h after FAK shRNA plasmids transfected into HSC(0.76±0.08 vs 2.31±0.24); (6) The ratio of TIMP-1/MMP-13 was down-regulated by FAK shRNA: The results showed that the ratio of TIMP-1/MMP-13 was obviously increased in FN group than that in Con group, P<0.05. There was no significant difference among FN group, Sofast group and HK group, P>0.05. Compared with the HK group, the ratio of TIMP-1/MMP-13 at mRNA level can be significantly down-regulated by 80.82% at 36 h after FAK shRNA treatment(0.42±0.03 vs 2.19±0.17), and the ratio of TIMP-1/MMP-13 at protein level can be significantly down-regulated by 79.15% at 48 h after FAK shRNA treatment (0.44±0.01 vs 2.11±0.05); (7) MT1-MMP mRNA and protein were up-regulated by FAK shRNA: real-time Q-PCR and Western blot results showed that the levels of MT1-MMP of FN group were significantly decreased when compared with that of Con group. Compared with the HK group, the expression of MT1-MMP mRNA can be significantly up-regulated by 65.88% at 36 h after FAK shRNA plasmids transfected into HSC(1.41±0.08 vs 0.85±0.04), and the expression of MT1-MMP protein can be significantly up-regulated by 62.26% at 48 h after FAK shRNA plasmids transfected into HSC(1.72±0.10 vs 1.06±0.12); (8) MMP-2 mRNA and protein were up-regulated by FAK shRNA: real-time Q-PCR and Western blot results showed that the levels of MMP-2 of FN group were significantly decreased when compared with that of Con group. Compared with the HK group, the expression of MMP-2 mRNA can be significantly up-regulated by 65.85% at 36 h after FAK shRNA plasmids transfected into HSC (1.36±0.06 vs 0.82±0.02), and the expression of MMP-2 protein can be significantly up-regulated by 66.25% at 48 h after FAK shRNA plasmids transfected into HSC (1.33±0.05 vs 0.80±0.05); (9) TIMP-2 mRNA and protein were down-regulated by FAK shRNA: real-time Q-PCR and Western blot results showed that the levels of TIMP-2 of FN group were significantly increased when compared with that of Con group. Compared with the HK group, the expression of TIMP-2 mRNA can be significantly down-regulated by 52.60% at 36 h after FAK shRNA plasmids transfected into HSC(0.73±0.03 vs 1.54±0.04), and the expression of TIMP-2 protein can be significantly down-regulated by 59.54% at 48 h after FAK shRNA plasmids transfected into HSC(0.53±0.08 vs 1.31±0.02); (10) The ratio of TIMP-2/MMP-2 was down-regulated by FAK shRNA: The results showed that the ratios of TIMP-2/MMP-2 at both levels were obviously increased in FN group than that in Con group, P<0.05. There was no significant difference among FN group, Sofast group and HK group, P>0.05. Compared with the HK group, the ratio of TIMP-2/MMP-2 at mRNA level can be significantly down-regulated by 71.28% at 36 h after FAK shRNA treatment(0.54±0.05 vs 1.88±0.08), the ratio of TIMP-2/MMP-2 at protein level can be significantly down-regulated by 75.61% at 48 h after FAK shRNA treatment (0.40±0.05 vs 1.64±0.13).Conclusions: FAK shRNA plasmids can be successfully transfected into HSC mediated by cationic polymer. The expression of FAK was inhibited specificly, and the expressions of CollagenⅠ, CollagenⅢwere also inhibited. This may be related to the up-regulation of MMP-13, MT1-MMP, MMP-2 and down-regulation of TIMP-1, TIMP-2. That is to say, FAK shRNA may inhibit the collagen synthesis by down-regulating the ratios of TIMP-1/MMP-13 and TIMP-2/MMP-2.