Fig. 2: MITF transcription factor controls miR-579-3p expression.

A Schematic illustration of the promoter region of ZFR/miR-579 gene showing the two MITF binding sites located −1182 bp and −361 bp upstream of the transcription start site (TSS). B Spearman’s correlation coefficient was calculated on 74 matched samples from the GSE54467 dataset, using miRNA/mRNA expression levels. C Quantification of miR-579-3p, MITF and TYR by using qRT–PCR in LOX IMVI, WM266 and M14 cell lines following 48 hours of transient transfection with scrambled (SCR) sequences or MITF siRNA. U6 and GAPDH were evaluated to normalize the results through ΔΔCt method. D Chromatin immunoprecipitation (ChIP) was performed on DNA extracted from LOX IMVI cells incubated with an anti-MITF antibody or with anti-mouse IgG, used as control. PCR analyses were used to evaluate MITF binding on ZFR/miR-579 or TYR promoter regions, using specific primers. Luciferase reporter assays of the constructs containing a region of 1000 bp with the two MITF binding sites in miR-579 promoter (E) (each point represent a biological replicate) or with the deletion of MITF binding sites (F) (Del1, Del2 or Double del) were used to test the capability of MITF to bind these regions. pGL3 plasmid (Basic) was used as control. Transient transfections of the above mentioned plasmids (500 ng each) have been performed in the presence or not of MITF siRNA or SCR for 48 hours. pLX313-Renilla plasmid (50 ng) has been used to normalize results. Student’s t test was performed to determine statistical significance *p < 0.05; **p < 0.01; and ***p < 0.001. qRT-PCR data are represented as mean (n = 3) ± SD; luciferase results are expressed as the mean of at least three independent experiments ±SEM.