Abstract:

Abstract:Objective  To establish human squamous carcinoma cell line Hep-2 stably overexpressing exogenous ANO1，and to  observe its effects on the biological characters of Hep-2 cells.Methods Primers were designed.DNA was extracted from clinical specimens of patients with head and neck squamous cell carcinoma, and taken as template for PCR.Gene sequences of ANO1 fragment were gotten.ANO1 fragment was inserted into the pSG-5 plasmid, which was transfected into Hep-2 cells.These Hep-2 cell lines with ANO1 stable overexpression were regarded as experiment group,and those Hep-2 cell lines transfected with blank plasmid were regarded as control group.The expression of ANO1 was detected by Western blotting and immunofluorescence assay in the Hep-2 cells after transfection.Boyden chamber invasion assay and adhesion assay were used to observe the effects of ANO1 overexpression on the biological characters of Hep-2 cells.Results The PSG-5 plasmid containing ANO1 fragments was successfully constructed.The expression of ANO1 in Hep-2 cell line after transfection was observed by Western blotting.The result of Western blotting showed that the target band occurred in each group, and 100 000 band was darker in experiment group.Indirect immunofluorescence assay showed that the bright green fluorescence presented in Hep-2 cells with stable overexpression of ANO1; while darker, lighter green fluorescence in Hep-2 cells after transfection with the blank plasmid.The percentage of ANO1 overexpressing Hep-2 cells migrating through the membrane and percentage of ANO1 overexpressing Hep-2 cells adhesion of cells were greater than those in  control group（P<0.05）.Conclusion  The Hep-2 cell line with ANO1 stable overexpression is successfully established.Stable ANO1 overexpression can improve the migration of Hep-2 cells.

Key words: head and neck neoplasms;anoctamin-1;Hep-2 cell