[Functional study of the eukaryotic fluorescent expression vector of truncated lymphoid enhancer-binding factor 1 (LEF-1) gene in SW480 cells].
OBJECTIVE
To construct a eukaryotic fluorescent expression vector of truncated lymphoid enhancer-binding factor 1 (LEF-1) gene and investigate its effect on the proliferation and apoptosis of human colonic carcinoma cell line SW480.


METHODS
Truncated LEF-1 gene was obtained by PCR and DNA recombination from human lymphoid node cDNA library. The PCR product of LEF-1 gene was inserted into the plasmid pMD-18T and sequenced. The truncated LEF-1 gene was inserted into the eukaryotic expression plasmid pcDNA3.1 and fused with mRFP for tracing. Using Lipofectamine 2000, the plasmid pcDNA3.1-LEF-1-mRFP was transfected into Hela cells and detected by Western blotting and fluorescence activated cell sorting (FACS). The changes in the growth, proliferation and apoptosis of the SW480 cells were observed after transfection with the plasmids.


RESULTS
The truncated LEF-1 gene was successfully cloned. After transfection with the plasmid pcDNA3.1-LEF-1-mRFP, the Hela cells expressed the product of LEF-1 as detected by Western blotting and FACS. The growth and proliferation of SW480 cells was inhibited and the cell apoptosis increased after transfection with the plasmid pcDNA3.1-LEF-1-mRFP, which also caused cell cycle arrest in G0/1 phase.


CONCLUSIONS
The eukaryotic expression fluorescent vector pcDNA3.1-LEF-1-mRFP has been constructed and expressed in eukaryotic cell line successfully. The truncated LEF-1 protein expressed in the transfected SW480 cells results in inhibition of the cell growth and proliferation with increased cell apoptosis and cell cycle arrest in G0/1 phase.
Wang
Shu-hong
SH
Department of Oncology, First Affiliated Hospital, Xi'an Jiaotong University College of Medicine, Xi'an 710061, China. wsh2003@126.com
Tian
Tao
T
Nan
Ke-jun
KJ
D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D005453:Fluorescence; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
C497573:LEF1 protein, human; D051821:Lymphoid Enhancer-Binding Factor 1; D011994:Recombinant Proteins