KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma

Abstract

Background: Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets.

Methods: Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy.

Results: We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration.

Conclusion: These functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets.

Keywords: Integrative analysis; KCNMA1; KCa1.1; Mesothelioma; Therapeutic targets; miR-17-5p; microRNA.

Fig. 1

Analysis pipeline. Differentially expressed gene lists in MPM from four public datasets (P < 0.05) were subjected to GSEA analysis using the C3 list in the Molecular Signatures Database (MSigDB v4.0) to identify enriched 3’UTR microRNA binding motifs [4]. The top 20 enriched microRNA motifs identified at P < 0.05 (False discovery rate adjusted) were considered significant and ranked between these four studies. Predicted target mRNAs from these enriched microRNA families were analyzed further using Gene Ontology (DAVID [68]) and Pathway Enrichment (Partek Genome Suite) to elucidate affected regulatory pathways. Confirmation of dysregulated candidate microRNA families in MPM cell lines (n = 7) and patient tumors (n = 59) were carried out using RT-qPCR. Furthermore, correlation of enriched microRNAs (downregulated) to gene expression of predicted targets (upregulated) were extracted based on our previous published array dataset [19]. Thus this analysis pipeline identifies and ranks candidate targets according to significance P value, correlation between miRNA-mRNA array data as well as being able to be targeted functionally by small molecule inhibitors

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)

Fig. 3

Molecular and pharmacological inhibition of KCa1.1 inhibits MPM cell line migration. a Transfection with miR-17-5p mimic or KCNMA1-specific siRNA (10 nM) resulted in significantly (* = P < 0.01) decreased KCNMA1 mRNA gene expression in MPM cell lines when compared with controls (Individual P values are included in Additional file 1: Table S5). RT-qPCR carried out as described for Fig. 2. b Immunofluorescent staining of KCa1.1 (Rabbit anti-KCNMA1, 1:500, Sigma) showed significant reduction in expression of KCa1.1 protein in MSTO cells transfected with miR-17-5p mimic or KCNMA1-specific siRNA (final concentration of 10 nM; bar = 400 μm). c Levels of KCNMA1 were measured following AGO2-IP using PCR and were higher in cells transfected with the miR-17 mimic. d Transfecting with miR-17 mimic did inhibit migration of mitotically inactivated H28 cells. Similar results obtained with other MPM cell lines are presented in Additional file 1: Figure S5. e In proliferation assays, the growth of MPM cell lines was inhibited by high concentrations of the KCa1.1 blocker paxilline. In contrast, a sub-lethal dose of paxilline (12.5 μM) inhibited migration (D, last 2 rows) and colony forming ability of MPM cells, plated at low density (f, histograms represent total dye in lysed colonies, as a percentage normalized to control, * P value all < 0.0001). P values for each comparisons are individually presented in the Additional file 1: Table S5. g Levels of cytosolic Ca²⁺ were estimated by overexpressing the soluble GCaMP5 Ca²⁺ reporter in MPM cells co-transfected with siRNA. Note, that the fluorescence intensity of the reporter is increased after the KCNMA1 knockdown, * P < 0.05, ANOVA with Holm-Sidak’s multiple comparisons test. h Analysis of the Ca²⁺ influx in MPM cells overexpressing the membrane-targeted LCK-GCaMP5 Ca²⁺ reporter and co-transfected with siRNA or miR-17-5p mimic. Calcium influx was induced at 5 s after the start of recording by application of 90 mM K⁺-containing buffer. Graphs show mean ± SEM fluorescence intensity of the reporter