Effects of wild-type PTEN overexpression and its mutation on F-actin in activated hepatic stellate cells
10.3760/cma.j.issn.1007-3418.2017.01.006
- VernacularTitle: PTEN过表达及其突变对活化肝星状细胞纤维状肌动蛋白的影响
- Author:
Lisen HAO
1
;
Yuling LIU
1
;
Guangling ZHANG
2
;
Jing CHEN
3
;
Xiaojie SONG
1
;
Yulan WANG
1
;
Jing WANG
1
;
Limin JIN
1
Author Information
1. Department of Gastroenterology, the Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei Province 063000, China
2. The Basic Medical College of North China University of Science and Technology, Tangshan Hebei Province 063000, China
3. The Life Science College of North China University of Science and Technology, Tangshan Hebei Province 063000, China
- Publication Type:Journal Article
- Keywords:
Hepatic stellate cells;
Cytoskeletal protein;
F-actin;
Calciumion concentration;
PTEN
- From:
Chinese Journal of Hepatology
2017;25(1):21-26
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro.
Methods:The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca2+ concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups.
Results:Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71, F = 1783.486, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca2+ compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17, F = 834.988, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05).
Conclusion:The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.
