Effects Of Adenovirus-mediated ShRNA Targeting PTEN On Cytoskeletal Protein Actin And Vinculin In Activated Hepatic Stellate Cells In Vitro

Objectives To investigate the effects of down-regulation of phosphatase and tensin homology detected on chromosome 10（PTEN）gene by adenovirus-mediated short hairpin RNA（sh RNA）on cytoskeletal protein actin and vinculin in activated hepatic stellate cells（HSC）in vitro.Methods The recombinant adenovirus Ad-sh RNA/PTEN carrying sh RNA targeting PTEN and expressing green fluorescent protein（GFP）,and the control adenovirus expressing GFP only were amplified by repeated infection with 293 A cells and its viral titer estimates were conducted.The activated rats HSC（HSC-T6）were cultured in vitro and transfected with sh RNA targeting PTEN via adenoviral vector.The expressions of PTEN protein and m RNA in HSC were measured by Western blot and Real-time fluorescent quantitation PCR（RT-q PCR）.Under the laser scanning confocal microscope（LSCM）,the cellular morphology,distribution of actin,and fluorescence intensity of filamentous actin（F-actin）,stress fibers and pseudopodium in activated HSC were examined by the phalloidin marked with tetramethylrhodamine isothiocyanate（TRITC）,while the vinculin changes in HSC were detected by immunofluorescence,and then the concentration of intracellular calcium ion(Ca²⁺)in HSC was detected by calcium fluorescent probe Rhod-2/AM.All the data were analyzed by SPSS17.0 statistical software.The measurement data were expressed as mean ± standard deviation（?）.One-way ANOVA was used to compare multipal groups,and LSD test was used between two groups.P<0.05 was considered statistically significant.Experimental grouping:（1）Control group,using DMEM instead of adenovirus for transfection.（2）Ad-GFP group,HSC were transfected with the empty adenovirus expressing GFP alone.（3）Ad-sh RNA/PTEN group,HSC were transfected with recombinant adenovirus Ad-sh RNA/PTEN.Results 1 Ad-GFP,Ad-sh RNA/PTEN were obtained by repeated infection of 293 A cells.The titers were 1.6′109 pfu/ml,1.3′109 pfu/ml,respectively.2 At 48 hours after adenovirus infection in HSC at Multiplicity of infection（MOI）100,the transfection rate of Ad-GFP group and Ad-sh RNA/PTEN group were measured by fluorescent microscopic counting.All of which were more than 80%.3 At 48 hours after adenoviral infection,the expression of PTEN in HSC was detected by real-time fluorescent quantitative PCR.Taking PTEN m RNA expression in control group as 1,then the PTEN m RN A expression in Ad-GFP group and Ad-sh RNA/PTEN group relative to control group were 0.92 times and 0.64 times.The expression levels of m RNA in Ad-sh RN A/PTEN group were significantly lower than those in control group and Ad-GFP group（P<0.05）,and there was no significant difference between control group and Ad-GFP group（P>0.05）.Western blot analysis recapitulated data by showing that the Ad-sh RNA/PTEN group（1.088±0.036）was significantly lower than those in control group（1.438±0.038）and Ad-GFP group（1.413±0.058）,P<0.05,while there was no significant difference between control group and Ad-GFP group（P>0.05）.4 The TRITC-labeled phalloidin assay showed,in control group and Ad-GFP group,the stress fibers were visible in HSC,with fiber short and sparse,arranged irregularly,furthermore,very few pseudopods formated in HSC.While at 48 h after HSC were infected by recombinant adenovirus carrying sh RN A targeting PTEN,the HSC showed starlike and stretched to the surrounding,the actin of the cells was closely arranged in order and had the increasing number,the pseudopodia fully extended outward,and stress fibers got larger and thicker.The fluorescence intensity of F-actin in Ad-sh RNA/PTEN group（1146.10±28.57）significantly enhanced（P<0.05）compared with control group（393.49±21.72）and Ad-GFP group（380.11±19.29）,while no significant difference between control group and Ad-GFP group was observed（P>0.05）.5 Detection of Ca²⁺ concentration in HSC by Calcium Fluorescence Probe Rhod-2/AM showed,intracellular Ca²⁺ concentration in Ad-sh RNA/PTEN group（1363.63±26.06）was statistically higher（P<0.05）than those in control group（334.62±16.97）and Ad-GFP group（348.05±19.99）,and no significant difference between the control group and Ad-GFP group was found（P>0.05）.6 Immunofluorescence assay showed vinculin expression,the fluorescence intensity（770.77±20.12）of HSC transfected with Ad-sh RNA/PTEN was significantly increased（P<0.05）compared with control group（381.13±9.12）and Ad-GFP group（383.87±12.15）.There was no significant difference between control group and Ad-GFP group（P>0.05）.Conclusions 1 Down-regulation of PTEN expression can remarkably promotes the formation of actin and enhances the reorganization of cytoskeleton in activated HSC in vitro.2 Down-regulation of PTEN expression can significantly increases the intracellular calcium ion concentration in activated HSC.3 Down-regulation of PTEN expression significantly up-regulate the expression of vinculin in activated HSC.