Fig. 3: miR-378a-5p (here abbreviated to miR-378) negatively regulates HOXD10 expression in melanoma cells.

qRT-PCR analysis of HOXD10 mRNA expression in M14 cells transfected with a mimic miRNA scramble control (mimic Ctrl), mimic miR-378 or miR-378 inhibitor (anti-miR-378) and the relative miRNA scramble control (anti-miR-Ctrl). The results are reported as fold induction ± SEM in cells transfected with mimic miR-378 or anti-miR-378 respect to relative controls. b Western blot analysis of HOXD10 protein expression in M14 cells transiently transfected with anti-miR-Ctrl or anti-miR-378. Representative images of one out of two independent experiments are reported. HSP72/73 was used as loading and transferring control. c Luciferase assay showing miR-378 binding to 3′UTR region of HOXD10 mRNA. Expression vectors carrying a luciferase reporter followed by the 3′-UTR regions of HOXD10 in their wild-type (wt, black bars) or mutated (mut, gray bars) forms, in the miR-378 complementary sequence were transfected in A375 cells in the presence of mimic Ctrl or mimic miR-378. Normalized luciferase activities of mimic miR-378 transfected cells respect to control were reported. a, c Statistical analysis was performed applying t-test. *p < 0.05, **p < 0.01. d Contingency table showing the distribution of high miR-378/low HOXD10 (N = 123, z score > 0) and low miR-378/high HOXD10 (N = 119, z score < 0) in primary (N = 57) and metastatic (N = 185) samples (p-value = 0.0001 using Fisher’s exact test). The high or low levels of miR-378 and HOXD10 expression were defined based on positive or negative z-scores of the miRNA\gene expression. In particular, z-score miR-378 > 0 & z-scores gene < 0 for high miR\low HOXD10; z-score miR-378 < 0 & z-score gene > 0 for low miR\high HOXD10.