| Hepatic fibrosis (HF) is the common pathological changes and necessary way of hepatic cirrhosis or liver cancer which caused by various chronic liver diseases. Hepatic stellate cells (HSCs), the main source of excessive extracellular matrix (ECM) in fibrotic liver tissue, play a vital role in the progress of fibrogenesis, and the central events in the liver fibrogenesis is the activation of HSCs and transformation into myofibroblast-like cells as well as the production of a large amount of collagen. Therefore, it is the key event to reverse HF that how to inhibite activation and proliferation or induce apoptosis of HSCs.Phosphatase and tensin homolog deleted on chromosome ten (PTEN), the first tumor-suppressing gene with phosphatase activity, can inhibit the proliferation and promote the apoptosis of tumor cells, and it is correlated with a variety of tumors.In recent years, PTEN research in cancer field has acquired significant progress, and the focus has been extended to nontumorous domain. Studies demonstrated that expression of PTEN protein or changes of its activity played an important role in organ fibrosis, and our previous study showed that the expression of PTEN protein and mRNA in fibrotic liver tissue with bile duct ligation were lower than that in the normal rats, and is negatively related to the activation and proliferation of HSCs in vivo, furthermore overexpression of PTEN can obviously inhibit the activation and proliferation of HSCs, induce its apoptosis in vitro. However, it remains unclear that the influence of blocking expression of PTEN on proliferation and apoptosis of HSC and intracellular signal transduction mechanism.RNA interference (RNAi) is the most effective gene silencing technology, which could specifically inhibit the transcription of target genes, and then reduce the level and function of the corresponding protein. Accordingly, the recombinant adenovirus PTEN shRNA, which carrys RNA interference sequences targeting PTEN, was constructed and transfered into activated rat HSC-T6 in vitro to block the expression of PTEN based on our previous study, and then the influence on proliferation and apoptosis of activated HSCs were observed to identify the role of PTEN on the regulation of HSCs biological behavior, and the mechanism that PTEN regulate proliferation and apoptosis of activated HSCs will be revealed from both sides, which might provide experimental and theoretical basis on seeking a new effective targets for the treatment of HF. Objective: To investigate the influence of proliferation and apoptosis of activated HSCs and the signaling mechanism after inhibition of PTEN expressionMethods: AD293T cells were used to amplify adenovirus vectors (PTEN shRNA, Ad-EGFP), and then virus titer was detected by fluorescence microscope. Ad-EGFP and PTEN shRNA were transfected into activated HSCs in vitro respectively. Experimental groups are as follows: (1) Control group, cells were cultured with DMEM containing 10% FBS under the regular conditions, except that DMEM containing no FBS and antibiotics was used in transfection step instead of adenovirus; (2) Ad-EGFP group, HSCs was transfected with adenovirus expressing enhanced green fluorescent protein (EGFP); (3) PTEN shRNA group, HSCs were infected with adenovirus which carried RNA interference sequences targeting PTEN and expressed EGFP.Direct cytometry was applied to draw growth curve of HSCs, and MTT assay was used to detect cell proliferation, as well TUNEL assay and flow cytometry (FCM) were used to determine cell apoptosis. The expressions of PTEN, Bax, Bcl-2, ERK1, p-ERK1, Akt and p-Akt in HSCs were measured by Western blot, and the mRNA expressions of Akt and ERK1 were detected by real-time Q-PCR.Results: (1) Adenoviral vectors (viral titers of Ad-EGFP and PTEN shRNA: 1.2×109 pfu/ml and 1.1×109 pfu/ml, respectively) for experiment required were obtained by repeating amplifications of virus in AD293T cells. (2) Real-time Q-PCR was used to detect the expressions of PTEN in HSCs at 72h after adenoviral infection. Relative quantitative method (2-△△Ct method) was applied to compare the levels of PTEN mRNA expression among groups. The levels of PTEN mRNA expression in Ad-EGFP and PTEN shRNA group were 0.93- fold, 0.63-fold compared to that in control group, respectively. It has been seen that the expression of PTEN mRNA was significantly lower than those in both control and Ad-EGFP group (P<0.05), and there were no significant differences between the control and Ad-EGFP group on the level of PTEN mRNA expression. Furthermore western blot analysis was used to measure the level of PTEN protein expression. It showed that the expression of PTEN protein in PTEN shRNA group (1.11±0.03) significantly decreased compared to those in control group (1.49±0.04) and Ad-EGFP group (1.48±0.02), P<0.05, as well no significant differences were observed between the control and Ad-EGFP group (P>0.05). It is suggested that PTEN shRNA could be successfully transfected into HSCs. (3) The results of direct cytometry showed that there were no significant differences among the three groups at 24h after adenoviral infection. PTEN shRNA group can promote the proliferation of HSCs compared to control group at 48h and 72h, as time increased its proliferation rates gradually rised, it is showed that growth rates in PTEN shRNA group was 22.5% higher than that in control group at 72h when the proliferation rates reached to Maximum, the proliferation rates between the control and Ad-EGFP group showed no notable differences at each time point. (4) The results of MTT assay showed that there were no notable differences in the proliferation of HSCs at 24 h after adenoviral infection between the Ad-EGFP and PTEN shRNA group(P>0.05). A precipitous time-dependent increase in proliferation was observed in PTEN shRNA group at 48 h and 72 h after adenoviral infection, and proliferation rates were 29.51%, 43.29% respectively compared with the control group (P<0.05). Moreover, there were no significant differences between control and Ad-EGFP group at each time point on A value (P>0.05). (4) TUNEL assay showed that the apoptotic rates of HSCs in PTEN shRNA group (2.94%± 0.31%) were notably lower than those in control group (5.17%±0.27%) and Ad-EGFP group (5.34%±0.43%) at 72 h after adenoviral infection, P<0.05, as well no great differences were found on the apoptotic rates of HSCs between control and Ad-EGFP group (P>0.05). (6) The apoptotic rates of HSCs by PI labeled FCM suggested that which of PTEN shRNA (1.26%±0.18%) decreased greatly compared to that of control group (3.28%±0.42%) and Ad-EGFP (2.95%±0.41%) at 72 h after adenoviral infection, P<0.05, furthermore, there were no notable differences between control and Ad-EGFP group in apoptotic rates of HSC (P>0.05). (7) Western blot analysis suggested the expression of Bax decreased significantly at 72 h after PTEN shRNA infection compared to control and Ad-EGFP group (0.98±0.04vs 1.29±0.03 and 1.30±0.04, respectively, P<0.05), and no significant differences were observed in the expression of Bax protein between control and Ad-EGFP group (P>0.05). In contrast, the expression of Bcl-2 protein in HSCs at 72 h after PTEN shRNA (1.46±0.06) infection heightened markedly compared with those in control group (1.08±0.04) and Ad-EGFP group (1.07±0.05), P<0.05, and there were no notable differences between control and Ad-EGFP group in the expression of Bcl-2 protein (P>0.05). (8) Western blot and real-time Q-PCR suggested there were no great differences among the three groups in the level of Akt mRNA and protein expression at 72 h after adenoviral infection (P>0.05), while Western blot analysis showed the expression of p-Akt (Thr308) protein in HSCs at 72 h after adenoviral infection increased greatly in PTEN shRNA group (1.59±0.03) compared to those in control group (1.21±0.03) and Ad-EGFP group (1.16±0.04), P<0.05. Moreover, no notable differences were found in the expressions of p-Akt (Thr308) protein between the control and Ad-EGFP group (P>0.05). (9) Western blot and Real time Q-PCR showed there were no significant differences among the three groups in the level of ERK1 mRNA and protein expression at 72 h after adenoviral infection (P>0.05), while Western blot analysis suggested the expression of p-ERK1 protein in HSCs at 72 h after adenoviral infection increased greatly in PTEN shRNA group (1.20±0.024) compared to those in control group (0.73±0.05) and Ad-EGFP group (0.81±0.04), P<0.05, furthermore, there were no significant differences between control and Ad-EGFP group in the expression of p-ERK1 protein (P>0.05).Conclusion: Adenovirus-mediated shRNA targeting PTEN could successfully transfected and expressed in activated HSCs in vitro, and could promote the proliferation and inhibit apoptosis of HSCs. Meanwhile the expression of apoptosis regulating gene Bax is decreased and Bcl-2 is increased in activated HSCs, as well the phosphorylation of Akt and ERK were promoted and then PI3K/Akt and ERK1/2 signal transduction pathways were activated, it is suggested that PI3K/Akt and ERK1/2 signal transduction pathways play a important role in regulating the the proliferation and apoptosis of HSCs. |
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