Effects Of PTEN Down-regulation By Rna Interference On Microtubule And Microfilament-binding Protein Cortactin And Filamin A In Activated Hepatic Stellate Cells In Vitro

Objectives To investigate the effects of phoshatase and tensin homology detected on chromosome Ten(PTEN)gene down-regulation by RNA interference on microtubule and microfilament-binding protein cortactin and filamin A in activated hepatic stellate cells(HSC)in vitro.Methods The control adenovirus expressing green fluorescent protein(GFP)only,and the recombinant adenovirus Ad-sh RNA/PTEN carrying RNA interference —— short hairpin RNA(sh RNA)targeting PTEN and expressing GFP were amplified by repeated infection with 293 T cells and the 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 m RNA in HSC were measured by real-time fluorescent quantitation PCR,and the expressions of PTEN protein in HSC were measured by Western blot.Through using laser scaning confocal microscope(LSCM),the expressions of ?-tubulin,?-tubulin and microfilament-binding protein cortactin and filamin A in the activated HSC were detected by immunofluorescence,and the integral optical density(IOD)analysis was conducted by Image-pro plus 6.0 software.Through using SPSS 17.0 statistical software,all exprerimental data were analyzed and expressed as mean ± standard deviation(x ±s).The comparisons among multiple groups were conducted by using one-way ANOVA,and LSD test was used for comparison between two groups.P < 0.05 was considered statistically significant.The experiments were divided into three groups: control group,replacing adenovirus with DMEM for HSC transfection.Ad-GFP group,HSC were transfected with empty adenovirus expressing GFP only.Adsh RNA/PTEN group,HSC were transfected with recombinant adenovirus Ad-sh RNA /PTEN.Results 1 The adenovirus Ad-GFP and Ad-shRNA/PTEN were obtained by repeated infection of 293 T cells.The titers were 1.6 x 108 pfu/ml and 1.4 x 108 pfu/ml,respectively.2 Through using fluorescent microscopic counting,the transfection rate of Ad-GFP group and Ad-sh RNA/PTEN group were found to be more than 80% at 48 hours after adenovirus infection in HSC at multiplicity of infection(MOI)100.3 At 48 hours after adenovirus infection,the m RNA expression of PTEN in HSC was detected by realtime fluorescent quantitation PCR.Taking PTEN m RNA expression in control group as 1,then the PTEN m RNA 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 PTEN m RNA in Ad-sh RNA/PTEN group were significantly lower than those in control group and Ad-GFP group(P < 0.05),and there was no statistical difference in the PTEN m RNA of HSC between control group and Ad-GFP group(P > 0.05).Western blot analysis recapitulated data by showing that the Ad-sh RNA/PTEN group(1.008±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 statistical difference in the PTEN protein of HSC between control group and Ad-GFP group(P > 0.05).4 Immunofluorescence assay showed ?-tubulin mainly distributed in the cytoplasm of HSC in three groups.In control group and Ad-GFP group,the ?-tubulin of HSC was radiate and ramified and uniformly distributed around the nucleus.And in Ad-sh RNA/PTEN group,the ?-tubulin of HSC gradually gathered towards the ends of cells and the microtubules slightly thickened.The fluorescence IOD of ?-tubulin in Ad-sh RNA/PTEN group(101495.17±5005.39)was significantly(P < 0.05)higher than that of control group(40803.00±2006.59)and Ad-GFP group(42302.50±2537.93),and there was no significant difference in fluorescence IOD of ?-tubulin between Ad-GFP group and control group(P>0.05).5 Immunofluorescence assay showed the ?-tubulin mainly distributed in the cytoplasm of HSC and there were punctiform aggregation of ?-tubulin among three groups and the punctiform aggregation of ?-tubulin was more obvious in Ad-sh RNA/PTEN group.But there were no changes of subcellular distribution among three groups.The fluorescence IOD of ?-tubulin in Adsh RNA/PTEN group(41310.83±1544.68)was significantly(P<0.05)higher than those of control group(20410.68±1815.53)and Ad-GFP group(20137.50±1469.49),and there was no significant difference in the fluorescence IOD of ?-tubulin between Ad-GFP group and control group(P>0.05).6 Immunofluorescence assay showed the cortactin mainly distributed in the cytoplasm of HSC in three groups,and there were no changes of subcellular distribution in all groups.The fluorescence IOD of cortactin in Adsh RNA/PTEN group(54688.50±2095.53)was significantly(P<0.05)higher than that of control group(22959.94±1710.42)and Ad-GFP group(22547.11±1588.72),and there was no significant difference in the fluorescence IOD of cortactin between Ad-GFP group and control group(P>0.05).7 Immunofluorescence assay showed the fluorescence IOD of filamin A in HSC have no significant difference among Ad-sh RNA/PTEN group,Ad-GFP group and control group(P>0.05).But there were the subcellular distribution changes of filamin A in HSC among three groups,the filamin A of HSC in Ad-sh RNA/PTEN group mostly distributed in cytoplasm,while the filamin A of HSC in Ad-GFP group and control group mainly located in the nucleus.The karyoplasmic ratio of filamin A in Adsh RNA/PTEN group(0.69±0.06)was significantly(P<0.05)lower than those of control group(1.13±0.03)and Ad-GFP group(1.12±0.04),and there was no significant difference in the karyoplasmic ratio of filamin A between Ad-GFP group and control group(P>0.05).Conclusions 1 The down-regulation of PTEN expression can enhance polymerization of microtubule and result in its disorderly arrangement in activated HSC in vitro.2 The down-regulation of PTEN expression can significantly increase the expressions of ?-tubulin and ?-tubulin in activated HSC in vitro.3 The down-regulation of PTEN expression raises the expression of microfilament-binding protein cortactin in activated HSC in vitro.4 The down-regulation of PTEN expression changes the subcellular distribution of microfilament binding protein filamin A,that is,filamin A translocates from nucleus to cytoplasmin in activated HSC in vitro.