The preliminary functional study of a Lgr5-harboring SW480 cell line

Abstract

Objective To construct the pIRES2-EGFP-Lgr5 recombinant expression vector,establish Lgr5 transient transfected SW480 cells,and investigate the effect of Lgr5 overexpression on the biological characteristic of colon cancer cells.Methods cDNA fragment encoding human Lgr5 gene was amplified from human colon cancer tissue by reverse transcription-PCR.After Lgr5 gene was dissected with the restriction endonucleases XhoI and BamHI,it was inserted into pIRES2-EGFP vector.The recombinant pIRES2-EGFP/Lgr5 was then tranfected into SW480 cells by lipofectamine 2000.The expression of Lgr5 in the transfected cells was identified by flow cytometric analysis,Western blot and immunohistochemistry.The proliferation and the ability of-packing into spheroids of the transfected cells in present and in the absence of cell matrix were detected by CCK-8 assay and hanging drops assay,while migTation of the cells was analyzed by the scratch test.Results The recombinant pIRES2-EGFP/Lgr5 expression vector was constructed and transient transfected SW480 cell line with Lgr5 overexpression was established.Colon cancer cells with Lgr5 overexpression grew faster,packed into more compact spheroids resistant to mechanical disruption and had a weaker ability to migrate compared to the cells infected control vector.Conclusion The research has provided solid experiment foundation for further studies in the biological characteristic of Lgr5 and its role in the development of colon cancer. Key words: Lgr5 ; Eukaryotic expressing vector ; Transfection ; SW480 cell line ; Biological characteristics ;