miR-181a/b Modulate Sensitivity and Resistance to BRAF-Inhibitors by Targeting Important Signalling Networks in Melanoma To understand the molecular mechanisms adopted by miR-181a/b to regulate the sensitivity and resistance to target therapies in melanoma, we performed a transcriptome profiling of melanoma BRAF-inhibitor sensitive and resistant cell lines

miR-181a/b Modulate Sensitivity and Resistance to BRAF-Inhibitors by Targeting Important Signalling Networks in Melanoma To understand the molecular mechanisms adopted by miR-181a/b to regulate the sensitivity and resistance to target therapies in melanoma, we performed a transcriptome profiling of melanoma BRAF-inhibitor sensitive and resistant cell lines. melanoma growth was inhibited in A375 and M14 resistant melanoma cells transfected with miR-181a/b mimics, while miR-181a/b depletion enhanced resistance in sensitive cell lines. Collectively, our study exhibited that miR-181a/b could reverse the resistance to BRAF inhibitors in dabrafenib resistant melanoma cell lines. In addition, miR-181a and -181b are strongly down-regulated in tumor samples from patients before and after the development of resistance to targeted therapies. Finally, melanoma tissues with high miR-181a and -181b expression offered favorable outcomes in terms of Progression Free Survival, suggesting that miR-181 is usually a clinically relevant candidate for therapeutic development or biomarker-based therapy selection. (Mitochondrial transcription factor A). Overexpression of miR-181a/b or inhibition of expression may thereby Cyclothiazide represent novel therapeutic approaches to increase the efficacy of BRAFi. Moreover, we propose that miR-181a/b expression levels may be a useful biomarker to improve patient selection for targeted therapy and for monitoring the onset of resistance. 2. Results 2.1. Melanoma Cells with Acquired Resistance to Dabrafenib Display Changes in miRNA Expression Pattern In order to identify miRNAs potentially involved in BRAF inhibitor resistance, we analyzed the miRNAome of melanoma cell lines sensitive to the BRAF inhibitor dabrafenib and their resistant counterparts. To explore this experimentally, we generated BRAF inhibitor-resistant A375 and M14 cells (A375-BIR and M14-BIR) by continuous selective culture. Using Small-RNAseq, a microRNA differential expression (DE) analysis on A375 and A375-BIR cells was performed to identify miRNAs regulating the resistance to Cyclothiazide dabrafenib treatment. Sixty-five miRNAs were found to have Cyclothiazide 1.5-fold or greater differences in levels in A375 cells in comparison to the resistant counterpart (Physique 1A and Table1) in both control (0.1% DMSO) and dabrafenib treated groups. CD164 The majority of differentially expressed miRNAs showed high-fold changes (greater than 2). Data are offered in Physique 1A as a heatmap showing differentially expressed (DE) miRNAs up-regulated (n = 27) and down-regulated (n = 38) in A375 sensitive cells with respect to A375-BIR. Open in a separate window Physique 1 (A) Hierarchical clustering analysis and warmth map based on the expression profiles of 65 miRNAs in responding and non-responding melanoma cells to dabrafenib treatment. Cluster analysis grouped samples and miRNAs according to similarity in expression. miRNAs are in rows and samples in columns. The miRNA clustering tree is usually indicated around the left and the sample clustering tree is at the top. Red color represents up-regulated expression and blue marks downregulated genes. Yellow indicates resistant cells and blue indicates responsive cells. miRNA, microRNA. To produce the heatmap we converted the read counts into log2-counts-per-million (logCPM) values. (B) Putative target genes of differentially expressed miRNAs Cyclothiazide were obtained from TargetScan and utilized for KEGG pathway enrichment analysis. Only pathways with an adjusted value 0.01 were considered and listed according to a decreasing value of the combined score. (C,D) The expression levels of miR-181a and b in A375, A375-BIR, M14 and M14-BIR melanoma cells, measured by quantitative Real-Time PCR (qRT-PCR). (E,F) The expression levels of miR-181a and b in A375, A375-BIR, M14 and M14-BIR melanoma cells, treated with 200 nM dabrafenib (DAB) and vehicle control (DMSO), measured by qRT-PCR. The miRNA expression levels were normalized to the internal control U6. **** 0.0001; ns, not significantly. The analysis of DE miRNAs allowed us to identify several miRNAs (e.g., in down-regulated group Cyclothiazide miR-126-3p, miR-146a-5p, miR-224-5p; in up-regulated group miR-424-3p, miR-503-5p, miR-509-3p) known to be involved in the onset and/or progression of melanoma and in resistance, thus confirming the quality of our approach and results [17,18,19,20,21] (Table 1). Table 1 Differentially Expressed microRNAs between A375 and A375-BIR melanoma cell lines. 0.001. (C,D) M14 and M14-BIR cells were transiently transfected with 40 nM of the miR-181a and -181b mimic or inhibitor or the appropriate controls and assayed for proliferation by the MTT assay. Data are expressed in terms.