Effects Of PTEN Gene Silencing On Cell Collagen Metabolism Of Activated Hepatic Stellate Cells In Vitro

Hepatic fibrosis is a repair reaction of hepatic disease caused by various factors. It's the channel for chronic hepatic disease developed into liver cirrhosis. Its main characteristics are the increased over synthesis and irregular deposition of extracellular matrix (ECM). At present, it is believed that hepatic stellate cells (HSC) are the original cell of liver extracellular matrix formation which can produce mass of ECM and cytokines with plenty of collagenⅠ,Ⅲto make liver fibrosis by migration, proliferation, expression of various signal-transducing protein.Phosphatase and tensin homology deleted on chromosome ten (PTEN) is the first tumor-suppressing gene found to exhibit phosphatase activity. It can not only regulate the cell cycle by signal transduction, influence the cell growth development and dissolution, but also regulate the cell migration and adhesion. In recent years, PTEN research has gradually extended beyond domain of cancer to other diseases states, which becomes the new research hotspot. Studies demonstrated that PTEN is related to fibrotic diseases. Our preliminary study showed that the expression of PTEN protein were lower than the normal rats in liver tissue of common bile duct ligation ones, negatively correlated with the in vivo HSC activation and proliferation. Overexpression of PTEN can significantly inhibit the activation of HSC proliferation, induce apoptosis, but absence of PTEN will regulate and control the activation of HSC proliferation and promote the phosphorylation of Akt and ERK.Up to now, RNA interference (RNAi) is the most effective gene silencing technology, which can efficiently inhibit the transcription of target genes, and in turn reduce the corresponding protein level and function. Therefore, the short hairpin RNA (shRNA) targeting PTEN, was constructed and transduced into activated rat HSC-T6 to inhibit the expression of PTEN, and the influence on collagen metabolism of activated HSC was observed to detect the regulation of PTEN on HSC collagen metabolism, which might provide theory evidence for antifibrotic gene therapy.Objective: To investigate the effects of short hairpin RNA targeting PTEN expression on HSC collagenⅠ, collagenⅢ, MMP-13, TIMP-1, MMP-2, TIMP-2 changes during this process.Methods: Amplifications of adenoviral vectors (Ad-EGFP, PTEN shRNA) were performed in AD293T cells and transfected into activated HSC-T6 in vitro. The experiment can be divided into 3 groups: (1) Control group, cells were cultured under the same conditions, except that DMEM containing no FBS and antibiotics was used in place of the adenovirus; (2) Ad-EGFP group, HSC were infected with adenovirus expressing enhanced green fluorescent protein (EGFP) alone; (3) PTEN shRNA group, transfect short hairpin RNA targeting PTEN interference recombinant and express the PTEN shRNA adenovirus recombinant of EGFP.Make use of fluorescence microscope, flow cytometer detection transfection efficiency; detect the expression of PTEN protein before and after transfection by the technology of Western blot; detect the situation of collagenⅠand collagenⅢexpression before and after transfection by the method of Immunocytochemistry; detect the expression of collagenⅠand collagenⅢ, MMP-13, TIMP-1, MMP-2, TIMP-2 protein before and after transfection by the technology of Western blot.Results:①Adenoviral vectors (viral titers of Ad-EGFP and PTEN shRNA: 1.2×109 pfu/ml, 1.1×109 pfu/ml, respectively) for experiment were obtained via performing repeated amplifications of virus in AD293T cells.②At 72h after adenoviral transfection, Western blot analysis showed that the expressions of PTEN protein in HSC in PTEN shRNA group (1.10±0.03) decreased significantly compared to those in control group (1.47±0.07) and Ad-EGFP group (1.38±0.08), P<0.01; Moreover, no significant difference was found in expressions of PTEN protein between control group and Ad-EGFP group (P>0.05).③At 72h after adenoviral transfection, immunocytochemical staining showed that collagenⅠand collagenⅢin PTEN shRNA group was significantly greater than the Control group and Ad-EGFP group.④Western blot technology verified that after the 72h adenoviral transfection, the expressions of collagenⅠprotein in PTEN shRNA group (0.41±0.01) increased significantly compared to those in control group (0.32±0.01) and Ad-EGFP group (0.33±0.01), P<0.01; moreover, no significant difference was found in expressions of collagenⅠprotein between control group and Ad-EGFP group (P>0.05); the expressions of collagenⅢprotein in PTEN shRNA group (0.38±0.03) increased significantly compared to those in control group (0.29±0.03) and Ad-EGFP group (0.29±0.04), P<0.05; moreover, no significant difference was found in expressions of PTEN protein between control group and Ad-EGFP group (P>0.05).⑤At 72h after adenoviral transfection, Western blot analysis showed that the expressions of MMP-13 protein in HSC in PTEN shRNA group (0.41±0.02) decreased significantly compared to those in control group (0.66±0.04) and Ad-EGFP group (0.71±0.05), P<0.01; Moreover, no significant difference was found in expressions of MMP-13 protein between control group and Ad-EGFP group (P>0.05); While the expressions of TIMP-1 protein in HSC in PTEN shRNA group (0.27±0.001) increased significantly compared to those in control group (0.23±0.01) and Ad-EGFP group (0.22±0.01), P<0.01; Moreover, no significant difference was found in expressions of TIMP-1 protein between control group and Ad-EGFP group (P>0.05).⑥At 72h after adenoviral transfection, Western blot analysis showed that the expressions of MMP-2 protein in HSC in PTEN shRNA group (0.29±0.01) decreased significantly compared to those in control group (0.34±0.01) and Ad-EGFP group (0.33±0.003), P<0.01; Moreover, no significant difference was found in expressions of MMP-2 protein between control group and Ad-EGFP group (P>0.05); While the expressions of TIMP-2 protein in HSC in PTEN shRNA group (1.26±0.03) increased significantly compared to those in control group (0.96±0.01) and Ad-EGFP group (0.93±0.01), P<0.05; Moreover, no significant difference was found in expressions of TIMP-2 protein between control group and Ad-EGFP group (P>0.05).Conclusion: PTEN shRNA and Ad-EGFP were successfully transfected into activated HSC in vitro; PTEN shRNA can inhibit the expression of PTEN protein,promote the synthesis of collagenⅠand collagenⅢ; PTEN shRNA can down the expression of MMP-13 and MMP-2, increase the expression of TIMP-1 and TIMP-2.