| Hepatic fibrosis is the common pathological changes of hepatic cirrhosis developed from varied kinds of chronic hepatic disease caused by many pathogenic factors. And the increased oversynthesis and irregular deposition of extracellular matrix (ECM) compose the main pathological characteristic of hepatic fibrosis. Currently, it is believed that most of fibroplasia derived fromhepatic stellate cells (HSC), the activation of which play a pivotal role in start of fibrosis process. What make the activated HSC appear to be the dominant driving force in fibrosis are the migration, proliferation, expression of various signal-transducing protein, and production of mass of ECM and cytokines with plenty of collagen fibers in advanced lesion of liver fibrosis. Along with the in-depth study in the pathogenesis of liver fibrosis, it has been an important strategy to inhibit the activation and proliferation of HSC which might help to derease ECM synthesis in blocking and reversion of hepatic fibrosis.Phosphatase and tensin homolog deleted on chromosome ten (PTEN), the first tumor-suppressing gene found to exhibit phosphatase activity, negatively regulates cell cycle, inhibits the proliferation and promotes the apoptosis of tumor cells. As such, it is reasonable to conclude that dysfunction or absence of PTEN is intimately related to the formation and progression of human tumors.Nevertheless, PTEN research has gradually extended beyond domain of cancer in recent years, the focus has been moved to other diseases states. Studies demonstrated that the absence of PTEN in specific hepatic cells may result not only in hepatocellular carcinoma, but also in non-alcoholic steatohepatitis, which was closely related to hepatic fibrosis. Our preliminary study showed that the expression of PTEN protein and mRNA 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 in vitro, and also negatively regulate the cell cycle progression of HSC by down-regulating the expressions of cyclinD1 and CDK4 as well as up-regulating the expression of P27kip1. However, it remains unclear that the influence of low expression or absence of PTEN on the cell cycle of activated HSC in vitro.Up to now, 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, the recombinant adenovirus Ad-siRNA/PTEN, which expressed 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 cell cycle of activated HSC was observed to detect the regulation of PTEN on HSC biological behavior. These data supported a possibility to interpret the regulatory mechanism of PTEN on HSC proliferation, which might make it a chance for the new targets in antifibrotic therapy.Objective: To investigate the effects of specific inhibition of PTEN expression on cell cycle of activated HSC and the signaling mechanism using transgenic technology.Methods: Amplifications of adenoviral vectors (Ad-SiRNA/PTEN, Ad-EGFP) were performed in AD293T cells and then the viral titer were detected. Ad-SiRNA/PTEN, Ad-EGFP were transfected into activated HSC in vitro, respectively. Cells were 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) Ad-siRNA/PTEN group, HSC were infected with adenovirus harboring genes for RNA interference against PTEN and EGFP.PI labed FCM was then used to detect cell cycle phase of activated HSC. And the expressions of PTEN, cyclinD1, cyclin dependent kinase 4 (CDK4), P27kip1, Akt, p-Akt (Thr308), ERK and p-ERK in HSC were measured by Western blot respectively.Results: (1) Adenoviral vectors (viral titers of Ad-siRNA/PTEN and Ad-EGFP: 1.1×109 pfu/ml, 1.2×109 pfu/ml, respectively) for experiment were obtained via performing repeated amplifications of virus in AD293T cells. (2) After infection by Ad-siRNA/PTEN Western blot results showed that the expressions of PTEN protein in HSC at different time points were significant different: at 0 h, 24 h, 48 h and 72 h , the expressions of PTEN protein were 2.46±0.06, 1.78±0.04, 1.43±0.12 and 0.79±0.14(P<0.01), respectively. At 72 h after adenoviral transfection, Western blot analysis showed that the expressions of PTEN protein in HSC in Ad-siRNA/PTEN group (0.43±0.02) decreased significantly compared to those in control group (1.23±0.17) and Ad-EGFP group (1.18±0.05), P<0.01; Moreover, no significant difference was found in expressions of PTEN protein between control group and Ad-EGFP group, P>0.05. (3) At 72 h after adenovirus transfection, Cell cycle phase of HSC in each group was detected by PI labed FCM. At G0/G1 phase, the number of HSC in Ad-siRNA/PTEN group (57.70%±4.37%) decreased greatly compared with those in control group (68.13%±1.00%) and Ad-EGFP group (69.57%±2.15%), P<0.01; At S phase, the number of HSC increased notably in Ad-siRNA/PTEN group (30.70%±5.13%) compared with control group (13.13%±1.78%) and Ad-EGFP group (12.37%±1.33%), P<0.01; At G2/M phase, the number of HSC in Ad-siRNA/PTEN group (11.60%±2.72%) decreased significantly compared to those in control group (18.73%±2.78%) and Ad-EGFP group (18.40%±2.79%), P<0.01; while there was no significant difference in the number of HSC between Control group and Ad-EGFP group (P>0.05). (4) At 72 h after adenoviral transfection, the expression of cyclinD1 protein in HSC was analyzed by Western blot, and it was greatly increased in Ad-siRNA/PTEN group (2.78±0.18), comparing to those in control group (1.93±0.12) and Ad-EGFP group (1.87±0.03), P<0.01; while no significant difference was observed between control group and Ad-EGFP group (P>0.05). (5) The expressions of CDK4 protein in Ad-siRNA/PTEN group (0.49±0.01), were markedly higher than those in control group (0.41±0.01) and Ad-EGFP group (0.39±0.02), P<0.01; while no significant difference was detected between control group and Ad-EGFP group (P>0.05). (6) The protein expressions of P27kip1 in HSC in Ad-siRNA/PTEN group (1.57±0.03) decreased significantly compared to those in control group (1.91±0.03) and Ad-EGFP group (1.95±0.02), P<0.01; Moreover, no significant difference was found in between control group and Ad-EGFP group (P>0.05). (7) The protein expressions of Akt in HSC in Ad-siRNA/PTEN group (2.71±0.22) decreased significantly compared to those in control group (3.4±0.16) and Ad-EGFP group (3.39±0.38), P<0.01; while no significant difference was found between control group and Ad-EGFP group (P>0.05). (8) The protein expressions of p-Akt(Thr308) in HSC in Ad-siRNA/PTEN group (2.27±0.02) increased significantly compared to those in control group (1.69±0.01) and Ad-EGFP group (1.74±0.01), P<0.01; and no significant difference was found between control group and Ad-EGFP group (P>0.05). (9) The protein expressions of ERK in HSC in Ad-siRNA/PTEN group (0.37±0.03) decreased compared to those in control group (0.48±0.04) and Ad-EGFP group (0.54±0.04), P<0.01; while no significant difference was found between control group and Ad-EGFP group (P>0.05). (10) The protein expressions of p-ERK in HSC in Ad-siRNA/PTEN group (4.47±0.08) increased significantly compared to those in control group (3.29±0.08) and Ad-EGFP group (3.36±0.4), P<0.01; moreover, no significant difference was found between control group and Ad-EGFP group (P>0.05).Conclusion: Ad-siRNA/PTEN and Ad-EGFP were successfully transfected into activated HSC in vitro. It was observed that PTEN significantly promoted the G1/S phase transformation in activated HSC in vitro. Besides it brought the up-regulation of cyclinD1 and CDK4 and the down-regulation of P27kip1 and accelerated the phosphorylation of Akt and ERK at translational levels in activated HSC in vitro, which may be one of the important mechanisms of Ad-siRNA/PTEN to promote HSC cell cycle progression.
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