Construction and functional study of three plasmids including Smad3 WT, Smad3 EPSM and Smad3 3S-A stably transfected HepG2 cell lines
10.3969/j.issn.1001-1978.2016.06.017
- VernacularTitle:S mad3 WT、S mad3 EPS M、S mad33 S-A 3种质粒稳转HepG2细胞株的建立与功能研究
- Author:
Jiajun WU
;
Yufeng JIANG
;
Chao WU
;
Ying MA
;
Ning CHEN
;
Lifenfang TAO
;
Yulu ZHANG
;
Xianglong ZHAO
;
Yan YANG
- Publication Type:Journal Article
- Keywords:
Smad3 WT;
Smad3 EPSM;
Smad3 3S-A;
HepG2 cells;
stable transfection;
cell proliferation;
cell cycle;
apoptosis
- From:
Chinese Pharmacological Bulletin
2016;32(6):825-831
- CountryChina
- Language:Chinese
-
Abstract:
Aim Toconstructthreeplasmidsincluding Smad3 WT,Smad3 EPSM and Smad3 3S-A stable transfection in HepG2 cell lines to investigate phospho-domains of Snad3(pSmad3C or pSmad3L),their pro-tein expression and roles in HepG2 cell proliferation, apoptosisandcellcycle.Methods Threeplasmidsin-cluding Smad3 WT (Carry the wild Smad3 gene ), Smad3 EPSM(Carry the mutated phosphorylation site in linker region of Smad3 gene)and Smad3 3S-A(Car-ry the mutated phosphorylation site in C-terminal of Smad3 gene)were respectively transfected into HepG2 cells by using a liposome transfection reagent.Verifi-cation of positive cells was done by screening with G418 via co-culture.Transfection efficiency was deter-mined by Western blot.Cell proliferation was induced by exogenous TGF-β1 in the respective stably transfect-ed HepG2 cell lines.Cell proliferation was monitored by MTT.Cell cycle and apoptosis were determined by flowcytometry(FCM).Results Therewaselevated protein expression of the respective phospho-domain sites in the stably transfected HepG2 cells for Smad3 WT(C-terminus and Linker),Smad3 EPSM(C-termi-nus)and Smad3 3S-A(Linker),which indicated suc-cessful stable transfection of HepG2 cell lines.The re-sults from MTT experiment showed that TGF-β1 could induce proliferation of HepG2 cells with or without the transfection of Smad3 WT,Smad3 EPSM and Smad3 3S-A plasmids,meanwhile transfected Smad3 EPSM plasmids could significantly inhibit proliferation of HepG2 cells induced by TGF-β1 , and transfected Smad3 3S-A plasmids accelerate proliferation of HepG2 cells induced by TGF-β1 .Cell cycle analysis showed that the G0/G1 phase of HepG2 cells with stable trans-fection of Smad3 EPSM plasmid increased compared with HepG2 cells with or without stable transfection of Smad3 WT plasmid,meanwhile the G2/M phase of HepG2 cells with stable transfection of Smad3 3 S-A plasmid increased.Compared with Smad3 WT trans-fected cells, apoptosis in Smad3 EPSM transfected cells was markedly increased,while that of Smad3 3S-Atransfectedcellsdecreased.Conclusions Thethree plasmids of Smad3 WT,Smad3 EPSM and Smad3 3S-A stably transfected HepG2 cell lines have been suc-cessively constructed.The construction of three plas-mids transfected HepG2 cell lines provides the research foundation for studying medical as well as possible reg-ulatory mechanism of pSmad3 C/pSmad3 L.
