Fig. 1

Knockdown of ADAM8 alters miRNA expression in TNBC cells. a Schematic representation of the various domains of the human ADAM8 protein and the different forms observed after processing in cancer cells. b, c MDA-MB-231 cells were transfected with 10 nM of either control siRNA (siCtrl) or the ADAM8 siRNAs siADAM8-1 or siADAM8-2 (siA8-1 or siA8-2). After 72 h, WCEs and RNA were isolated. WCEs (25 μg) were examined by Western blotting for expression of ADAM8, and β-tubulin as a loading control. A representative blot is shown (n = 3). ADAM8 forms and molecular weight (MW) markers are as indicated (b). RNAs isolated from siCtrl and siADAM8-2 cells were subjected to an RT-qPCR-based miRNA array assay and the resulting heat map representation of miRNA expression displaying a greater than twofold change presented as fold-changes in siADAM8-2 compared to siCtrl in two independent experiments (Arrays 1 and 2) (c). The legend for the fold-changes in the heat map is given to the left. d RNA from MDA-MB-231 cells transfected with 10 nM of siCtrl, siADAM8-1 or siADAM8-2 for 72 h were subjected to RT-qPCR for the indicated miRNAs. siCtrl was set to 1 and the fold changes given as mean ± SD from three independent experiments. e WCEs from SUM-149 cells transfected with 10 nM of siCtrl, siADAM8-1 or siADAM8-2 for 72 h were subjected to Western blotting for expression of ADAM8, and β-actin as a loading control. A representative blot is shown (n = 3). Positions of the proform, active and remnant forms of ADAM8 and MW markers are as indicated. f RNAs were isolated from SUM-149 cells transfected, as in part e, and samples subjected to RT-qPCR for miR-720 levels. siCtrl was set to 1 and relative (Rel.) levels of miR-720 are given (mean ± SD from three independent experiments). *P < 0.05, Student’s t test. CRD cysteine-rich domain, CTD cytoplasmic tail domain, DI disintegrin, ELD epidermal growth factor-like domain, MP metalloproteinase, PRO prodomain, and TD transmembrane domain.