The B-Raf^V600E-Twist axis regulates miR-10b expression in melanoma.

(a) Twist1 expression positively correlates with B-RafV600E mutation status in multiple cell lines. qRT- PCR shows a strong positive correlation of Twist1 expression with B-RafV600E or N-Ras activating mutations in a panel of melanoma cell lines. (b-c) miR-10b displays significant correlation with Twist1 mRNA expression in B-RafV600E mutant melanoma. Scatter plots showing correlation of miR-10b expression with (b) Twist1 mRNA expression and (c) Myc mRNA expression in B-RafWT and B-RafV600E samples from 407 TCGA-SKCM samples. r denotes Pearson correlation coefficient. p refers to a Pearson correlation P-value. (d) Upper panel: Box plots display miR-10b expression distribution in B-RafWT and B-RafV600E from 201 TCGA-SKCM samples where Twist1 mRNA expression was high. The median expression value for the wild-type samples was 50,343, and for the mutant was 66,002. The p-value for a Mann-Whitney test was 0.0149. Lower panel: Box plots display miR-10b expression distribution in BRafWT and BRafV600E from 206 TCGA-SKCM samples where Twist1 mRNA expression was low. The median expression value for the wild-type samples was 69,160, and for the mutant was 54,373. The p-value for a Mann-Whitney test was 0.02. (e) Protein expression of B-Raf and Twist1 in sk-mel-28 or Mel505 cell lines expressing siRNAs against B-Raf or control siRNAs (siGFP or siCON). Relative levels of B-Raf or Twist1 are indicated. (f) Upper panel, relative Twist1 mRNA expression in sk-mel-24 cells expressing three different Twist1 specific siRNAs or siGFP control as assessed by qRT-PCR. Lower panel, relative mR-10b expression in the indicated siRNA expressing sk-mel-24 cells as assessed by TaqMan qRT-PCR. (g) ChIP analysis on M505 cells with or without the ectopic expression of B-RafV600E. The putative E-boxes in miR-10b promoter are shown as red boxes. The locations of primers used for ChIP assays are marked by black arrows (amplicons). ChIP analysis was conducted using specific antibodies that recognize human Twist1, as well as control IgG.