Résumé
Objective To investigate the inhibition of lymphoid enhancer factor-1 (LEF-1) expression in human malignant melanoma cell line A375 by RNA interference method. Methods Sense and antisense oligonucleotides with hairpin structures, targeted specifically at LEF-1 mRNA, were designed, synthesized, then linked to the expression vector psilencer3.1-H1 neo after annealing. After identification, the re-combinant psilencer3.1-H1/LEF-1 siRNA was used to transfect the cultured A375 cells by a liposome-medi-ated method. The cells expressing the recombinant RNA was detected by G418 screening. The mRNA and protein levels were detected by RT-PCR, Western blotting and immunocytochemistry, respectively. Results The expression vector psilencer3.1-H1/LEF-1 siRNA was successfully constructed, and its stable expression in cell clones was achieved. The mRNA and protein levels of LEF-1 were both down-regulated in the trans-fected cells. Conclusion The recombinant of psilencer3.1-HI/LEF-1 siRNA can inhibit the mRNA and protein expression of LEF-1 in A375 cells.
Résumé
Objective To study the effects of ?-catenin-dependent lymphoid enhancer factor(LEF-1) isoforms on biological behavior of HeLa cells.Methods ?-catenin-dependent LEF-1 genes were obtained by PCR from human lymphoid node cDNA library and inserted into pcDNA3.1/V5-His vector to construct the eukaryotic expression plasmid pcDNA3.1-F-LEF-1.Using lipofectamineTM 2000,the plasmid pcDNA3.1-F-LEF-1 was transfected into Hela cells.Then we screened the stable cell lines that expressed the truncated LEF-1 isoforms by G418 and identified the expression of target gene with Western blot.Then we analyzed the proliferation,apoptosis,cell clone formation and capability of tumor formation in vivo of transfected cell lines.Results We successfully constructed the ?-catenin-dependent LEF-1 eukaryotic expression plasmid and obtained the stable HeLa cell lines that expressed the full-length LEF-1 isoforms.The proliferation and capability of tumor formation in vivo of transfected cells were increased while apoptosis was decreased.Conclusion The overexpression of ?-catenin-dependent isoforms can stimulate the malignant biological behavior of HeLa cells.