Knockdown of Egr1 decreases HGF-mediated invasion and activation of MMP2, MMP9, and expression of MT1-MMP.

SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in Materials and Methods. Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline (7A). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF (7B, 7C). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF (7D, 7E). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells (7F). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.