Effects Of Focal Adhesion Kinase Gene Silencing On Proliferation And Apoptosis Of Hepatic Stellate Cells

Hepatic fibrosis (HF), which may develop into liver cirrhosis at the end stage, is the common pathological changes of a variety of chronic liver diseases. The activation, proliferation of hepatic stellate cells (HSCs), which are the major source of excessive extracellular matrix (ECM) in fibrotic liver tissue, are the central events in the liver fibrogenesis. Therefore, inhibition of activation and proliferation or induction of apoptosis in HSCs is the key event to reverse HF.Focal adhesion kinase (FAK) is a critical mediator that connects integrin and the signal molecules of downstream in integrin-signal transduction pathway, and is the convergence of many signal pathways. Activation of FAK can regulate the signal molecules of downstream and induce the reorganization of cytoskeleton and the expression of some genes. For example, it can cause cascade reaction by Ras-dependent mitogen-activated protein kinase (MAPK) pathway to participate in many cellular behaviors,such as cell spreading, proliferation, differentiation and apoptosis.FAK is the key signaling molecule in integrin signal pathway. A number of studies show that FAK plays a key role in the proliferation and apoptosis in the fibroblasts, smooth muscle cells, endothelial cells and tumor cells, but it is not yet in-depth research in HSCs. RNA interference (RNAi) is the most effective gene silencing technology, which can specifically inhibit the transcription of target genes, and in turn reduce the level and function of the corresponding protein.Therefore, using RNAi technology, we choose FAK as the target gene and set about our research from the proliferation and apoptosis of HSCs. The recombinant plasmids targeting FAK can be successfully constructed by using a U6 promoter and enhanced green fluorescent protein (EGFP) vector, and can be successfully transfected into HSCs in preliminary work. Here we transfected FAK shRNA plasmid into HSCs mediated by cationic polymer, in order to observe the effects of FAK gene silencing on proliferation and apoptosis in fibronectin (FN)-stimulated HSCs and its mechanism.Objective: FAK shRNA plasmid was transfected into HSCs in order to observe the effects of FAK shRNA plasmid on proliferation and apoptosis in FN-stimulated HSCs and the roles of bcl-2, bax and signal transduction pathway mediated by FAK in the process.Methods: HSCs were cultured in vitro and FAK shRNA plasmid was transfected into FN-stimulated HSCs mediated by cationic polymer. 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 (shRNA). The concentration of FN is 10μg·mL^-1 from group (2) to group (5). The effect of FAK shRNA on the proliferation of HSCs was detected by MTT assay. The effect of FAK shRNA on the apoptosis of HSCs was examined by flow cytometry (FCM),terminal deoxynucleotidy1 transferase-mediated dUTP nick end labeling (TUNEL) and transmission electron microscope. And the levels of FAK, ERK₁, bcl-2, bax and caspase-3 in HSCs were assayed by real-time Q-PCR on mRNA level and Western blot on protein level, respectively.Results:â‘´FAK shRNA inhibited the expression of FAK mRNA: real-time Q-PCR results showed that the expression of FAK mRNA in FN group was obviously increased by 57.00% than that in Con group, P<0.01, but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the expression of FAK mRNA in FAK shRNA plasmid group was significantly reduced, P<0.01, and the highest down-regulated rate was 76.73% at 24 h after transfection;⑵FAK shRNA inhibited the expression and phosphorylation of FAK protein: Western blot results showed that the expression of FAK protein in FN group was obviously increased by 49.70% than that in Con group, P<0.01, but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the expression of FAK protein in FAK shRNA plasmid group was significantly reduced, P<0.01, and the highest down-regulated rate was 72.53% at 48 h after transfection. p-FAK (Tyr³⁹⁷) protein expression also showed the similar tendency. Western blot analysis results displayed that the expression of p-FAK protein was down-regulated after FAK shRNA transfection, and the highest down-regulated rate appeared at 48 h after transfection;⑶FAK shRNA inhibited the expression of ERK1 mRNA: real-time Q-PCR results showed that, the expression of ERK1 mRNA in FAK shRNA plasmid group was significantly reduced compared with the HK group, P<0.01, and the maximum reduction rate was 55.67% which appeared at 48 h after transfection;â‘·FAK shRNA inhibited the expression of ERK1, p-ERK protein: Western blot analysis showed that the expression of ERK1 protein was up-regulated after FN stimulation and down-regulated after FAK shRNA transfection, and the expression of ERK1 protein was reduced by 65.13% at 48 h after transfection. At the same time, the changes in p-ERK also showed similar tendency, the highest down-regulated rate was 75.47%, which appeared at 72 h after transfection;⑸FAK shRNA inhibited the proliferation of HSCs: Compared with the HK group, the proliferation of HSCs in FAK shRNA plasmid group was significantly inhibited, P<0.01, and the inhibition rates at 12 h, 24 h and 48 h after transfection were 11.08%, 15.12% and 28.62%, respectively. Herein, FAK shRNA could dramatically inhibit the proliferation of HSCs in a time -dependent manner;⑹FAK shRNA promoted the apoptosis of HSCs: the apoptotic rates of HSCs were assayed by FCM at 24 h after transfection. The apoptotic rate of HSCs in FN group was obviously lowered by 48.73% than that in Con group, P<0.01; but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the apoptotic rate of HSCs in FAK shRNA plasmid group was significantly increased (8.29%±0.79% vs 2.70%±0.31%), P<0.01. At the same time, the apoptotic rate of HSCs by TUNEL assay also showed similar tendency. The apoptotic rate of HSCs in FN group was obviously lowered by 52.01% than that in Con group, P<0.01, but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the apoptotic rate of HSCs in FAK shRNA plasmid group was significantly increased (19.00%±0.92% vs 7.63%±0.70%), P<0.01. HSCs in FAK shRNA plasmid group were observed by transmission electron microscopy, finding that the cellular membrane shrinkaged, nuclear chromatins agglutinated, shrunk and aggregated along inside of nuclear membrane forming of ball, petal or crescent shape. Sometimes, apoptotic bodies formed;⑺The expressions of caspase-3 mRNA and protein were increased: real-time Q-PCR results showed that the expression of caspase-3 mRNA in FN group was obviously reduced by 35.00% than that in Con group, P<0.05; but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the expression of caspase-3 mRNA in FAK shRNA plasmid group was significantly increased, P<0.01, and the highest up-regulated rate was 60.71% at 36 h after transfection. Western blot results showed that the expression of caspase-3 protein in FN group was obviously reduced by 20.87% than that in Con group, P<0.05; but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the expression of caspase-3 protein in FAK shRNA plasmid group was significantly increased, P<0.01, and the highest up-regulated rate was 75.29% at 48 h after transfection;â‘»bcl-2/bax ratio lowered: real-time Q-PCR results showed that the expression of bax mRNA in FN group was obviously reduced by 33.00% than that in Con group, P<0.01; but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the expression of bax mRNA in FAK shRNA plasmid group was significantly increased, P<0.01, and the highest up-regulated rate was 65.82% at 36 h after transfection. Western blot results showed that the expression of bax protein in FN group was obviously reduced by 19.08% than that in Con group, P<0.01; but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the expression of bax protein in FAK shRNA plasmid group was significantly increased, P<0.01, and the highest up-regulated rate was 66.06% at 48 h after transfection. Real-time Q-PCR results showed that the expression of bcl-2 mRNA in FN group was obviously increased by 35.00% than that in Con group, P<0.01; but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the expression of bcl-2 mRNA in FAK shRNA plasmid group was significantly reduced, P<0.01, and the highest down-regulated rate was 48.03% at 36 h after transfection. Western blot results showed that the expression of bcl-2 protein in FN group was obviously increased by 69.53% than that in Con group, P<0.01; but there was no obvious difference among FN group, sofast group and HK group, P>0.05. Compared with the HK group, the expression of bcl-2 protein in FAK shRNA plasmid group was significantly reduced, P<0.01, and the highest down-regulated rate was 71.90% at 48 h after transfection. Put the expressions of bcl-2 and bax at the transcription and translation levels in each group separately for one ratio, and the results showed that at the translation and transcription levels, FAK shRNA plasmid transfection into HSCs decreased bcl-2/bax ratio.Conclusions: FN can effectively stimulate the expression and phosphorylation of FAK and ERK1. After transient transfection of shRNA plasmid targeting FAK mediated by cationic polymer, FAK expression was reduced both in the levels of transcription and translation, with the decline of FAK phosphorylation. ERK1 expression also showed similar changes. FAK shRNA can inhibit the proliferation of HSCs in a time-dependent manner and induce the apoptosis of HSCs. Perhaps FAK-ERK signal transduction pathway and bcl-2/bax were involved in these processes.