Fig. 2
Identification of enriched microRNA families. a The top 20 enriched microRNA binding motifs in each MPM gene expression study were compared and the overlap between studies identified. Families enriched in more than one dataset are included in the table (see Additional file 1: Table S1 for top 20 enriched families in each dataset). RT-qPCR confirmed decreased expression of the miR-17 family in (b) MPM tumors (n = 59) compared with normal pleural tissue (n = 22) and (c) MPM cells lines compared with MeT-5A. The formalin-fixed paraffin embedded (FFPE) tumor tissues used in this study were described previously [62]. Total RNA was extracted from cell lines, tumors and normal pleura and used as template in RT-qPCR using microRNA-specific TaqMan assays (Additional file 1: Table S3) as previously described [17, 18]. Relative expression levels were calculated using the 2-ΔΔCq method [63] relative to MeT-5A or normal pleura. d Analysis of the top four enriched pathways related to the miR-17 family identified a number of target genes involved in multiple pathways. e Key miR-17 family target genes are coordinately regulated in signaling pathways contributing to MPM cell migration. Blue denotes upregulation > 1.5 fold, Yellow denotes downregulation < 1.5 fold. White arrow denotes direction in change of expression using data from Wright et al. [19]. f Expression analysis identified 40 predicted targets of miR-17 that were differentially expressed between MPM cells and MeT-5A cells; 20 of these targets were upregulated, including KCNMA1 and RT-qPCR confirmed upregulation of KCNMA1 in MPM cell lines (g). In a second series of tumor samples consisting of fresh-frozen samples from extrapleural pneumonectomy (EPP) patients, KCNMA1 was upregulated (h) and miR-17-5p downregulated (i) compared with normal pleural tissue controls (see Additional file 1: Table S4 for patient characteristics). j KCa1.1 expression in MPM tumor samples were analyzed by immunofluorescence microscope (Objective 40×, Axio imager.M2) showed high level of KCal.1 expression (right) of tumor area and low to no KCa1.1 expression of the non-tumor area (left)