Influences Of PTEN Down-regulation By RNA Interference On FAK/p130Cas And Paxillin Signal Transduction In Activated Hepatic Stellate Cells In Vitro

Objectives To investigate effects of the down-regulation of phosphatase and tensin homology deleted on chromosome Ten（PTEN）by adenovirus mediated short hairpin RNA（sh RNA）on focal adhesion kinase（FAK）/ p130Crk-associated substrate（p130Cas）and paxillin signal transduction in activated hepatic stellate cells（HSC）in vitro.Methods The recombinant adenovirus（Ad-sh RNA/PTEN）with sh RNA targeting PTEN and expressing green fluorescent protein（GFP）and empty vector（Ad-GFP）only expressing GFP were amplified in AD293 cells,and viral titer estimates were conducted.The sh RNA targeting to PTEN was transduced into the rats activated HSC（HSC-T6）in vitro via adenoviral vector.The protein expressions of PTEN,FAK,phosphorylated FAK(Thr³⁹⁷)[p-FAK(Tyr³⁹⁷)],p130 Cas and paxillin in activated HSC were analysised by Western blot.And the m RNA expressions of PTEN,FAK,p130 Cas and paxillin were analysised by real-time fluorescent quantitation PCR.All data were put into a database in Excel and were analyzed by statistical software of SPSS 21.0 and the measurement data was demonstrated as mean±standard deviation（x ±s）.Additionally,single-factor analysis of variance method（one-way ANOVA）was utilized to compare more groups,and LSD test was applied to inspect every two groups.If the possibility rates was less than 0.05,then which was regarded as statistical significance.Cells were grouped as follows:（1）Control group,HSC were cultured simply in DMEM with 8% FBS,and adenoviral was replaced by DMEM without FBS or antibacterial at transfection virus steps.（2）Ad-GFP group,HSC were infected with adenovirus expressing GFP alone.（3）Ad-sh RAN/PTEN group,HSC were infected with adenovirus carrying the sh RNA targeting PTEN and expressing GFP meanwhile.Results 1.Through the method of repeated infection in AD293 cell for obtaining experimental adenovirus,the viral titers of Ad-GFP and Ad-sh RNA/PTEN were 1.8×10⁹ pfu/ml and 1.4×10⁹ pfu/ml respectively.2.At 48 hours after adenovirus infection in HSC at Multiplicity of infection（M.O.I）100,the transfection efficiency of Ad-GFP and Adsh RNA/PTEN were checked by fluorescent microscopic counting,all of which were more than 80%.3.At 48 hours after HSC were infected by adenovirus,the PTEN m RNA expressions of HSC were detected by real time fluorescent quantitative PCR.The PTEN m RNA expressions quantity in control group was considered as 1,then PTEN m RNA expression in Ad-GFP group,Ad–sh RNA/PTEN group were 1.01 times,0.54 times,respectively,compared to control group.Obviously,PTEN m RNA expressions in Ad–sh RNA/PTEN group were significantly（P﹤0.05）lower than those in control group and Ad-GFP group,and no differences between control group and Ad-GFP group were observed,P﹥0.05.The protein expressions of PTEN in HSC were analysised by Western blot,Ad-sh RNA/PTEN group（0.19 ± 0.04）was remarkablely（P﹤0.05）lower than those in control group（0.44 ± 0.02）and Ad-GFP group（0.36 ± 0.05）,nevertheless the differences between control group and Ad-GFP group were no statistical significance（P>0.05）.4.Used western blot and real-time fluorescent quantitative PCR to detect FAK m RNA and protein expressions in HSC at 48 hours after adenovirus infection.The result showed that FAK m RNA and protein expressions in each group had no obvious changes（P>0.05）.5.The protein expressions of p-FAK(Tyr³⁹⁷)in HSC were tested by using Western blot,Ad-sh RNA/PTEN group（0.77±0.08）notablly increased,compared to the control group（0.47±0.05）and Ad-GFP group（0.44±0.02）,P<0.05.But there were no significant differences between control group and Ad-GFP group（P>0.05）.6.When HSC were infected by adenovirus for 48 hours,real time fluorescent quantitative PCR was used to test p130 Cas m RNA expressions of HSC.The m RNA expression of P130 Cas in the control group was 1,the Ad-GFP group and the Ad-sh RNA/PTEN group were 1.01 times,1.52 times,respectively.The p130 Cas m RNA expressions in Ad-sh RNA/PTEN group significantly（P<0.05）increased,compared to control group and Ad-GFP group.But no significant differences were observed in the expressions of p130 Cas m RNA between control group and Ad-GFP group.Applied western blot to detect p130 Cas protein expressions of HSC,the Ad-sh RNA/PTEN group（0.93±0.08）was notablly higher than those in control group（0.74 ± 0.07）and Ad-GFP group（0.72 ± 0.02）,P<0.05.But trere were no significant differences between the control group and the Ad-GFP group（P>0.05）.7.When HSC were infected by adenovirus for 48 hours,real time fluorescent quantitative PCR was used to detect the paxillin m RNA expressions.As the m RNA expressions of paxillin in the control group was 1,then the paxillin m RNA expressions in Ad-GFP group and Ad-sh RNA/PTEN group were 0.97 times,1.58 times,respectively.The paxillin m RNA expressions in Ad-sh RNA/PTEN group significantly（P<0.05）increased,compard with control group and Ad-GFP group,there were no significant differences between control group and Ad-GFP group（P>0.05）.Applied western blot technology to detect paxillin protein expressions of HSC,Ad-sh RNA/ PTEN group（0.91±0.05）was notablly higher than those in the control group（0.46±0.03）and Ad-GFP group（0.50±0.04）,which existed striking difference in statistic analysis（P<0.05）,nevertheless with no sharp differences between the control group and the Ad-GFP group（P>0.05）.Conclusions 1 The down-regulation of PTEN can enhance FAK signal transduction by promoting phosphorylation of FAK in activated HSC in vitro.2 The down-regulation of PTEN can strengthen the activity of FAK/p130 Cas signal transduction in activated HSC in vitro.3 The down-regulation of PTEN can highten the activity of paxilin signal transduction in activated HSC in vitro.