Figure 6

miR-125b-5p antagonizes pro-survival effect of BM milieu. (a) Quantitative RT-PCR of miR-125b-5p expression in NCI-H929, KMS-12-BM, INA-6, SK-MM-1 and U266 cells cultured in the presence or absence of either IL-6 (2.5 ng/ml) or IGF-1 (100 μg/ml) or HGF (150 μg/ml) (48-h time point). Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method. miR-125b-5p expression levels in cells cultured in the absence of growth factors were set as an internal reference. (b) Quantitative RT-PCR of miR-125b-5p expression in SK-MM-1 and U266 cells exposed to either DMSO or JQ1 (1 μM) or 10058-F4 (100 μM) (48-h time point). Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as ΔΔCt values calculated using the comparative cross threshold method. miR-125b-5p expression levels in cells exposed to DMSO were set as an internal reference. (c) Quantitative RT-PCR of miR-125b-5p expression in SK-MM-1 cells cultured in the presence or absence of either IL-6 (2.5 ng/ml) or IGF-1 (100 μg/ml) or HGF (150 μg/ml) and exposed to either DMSO or JQ1 (1 μM) or 10058-F4 (100 μM) (48-h time point). Raw Ct values were normalized to RNU44 housekeeping snoRNA and expressed as percentage of ΔΔCt values calculated using the comparative cross threshold method. miR-125b-5p expression level in cells cultured in absence of growth factors and exposed to DMSO was set as an internal reference. (d) CCK-8 assay was performed 2 days after transfection of INA-6 and NCI-H929 cells with miR-125b-5p or miR-NC. IL-6 (2,5 ng/ml) or IGF-1 (100 ng/ml) or HGF (150 ng/ml) were added to complete culture medium. (e) 7-AAD flow cytometry assay of INA-6 and NCI-H929 cells cultured in the presence or absence of HS-5/GFP+ stromal cell line. The assay was performed 48 h after transfection of MM cells with miR-125b-5p mimics or miR-NC. To discriminate between MM cells and HS-5 stromal cells, GFP negative cells were gated. (f) Flow cytometry analysis of IL6-R/CD126 expression on cell surface of INA-6 cells after transfection with miR-125b-5p or miR-NC (48-h time point). (g) Western blot analysis of total STAT3 (tSTAT3) and phosphorylated STAT3 (pSTAT3) in lysates from INA-6 cells transfected with miR-125b-5p or miR-NC (48-h time point). GAPDH was used as protein loading control.