Figure 1

miR-223 was up-regulated in breast cancer cell lines co-cultured with IL-4-activated macrophages. (A) miRNA profiling in macrophages and the breast cancer cell line SKBR3 using the DiscovArray miRNA array. Three of the miRNAs that were present at high levels in macrophages but absent in SKBR3 cells are shown. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages). (B) Quantitative real-time PCR (qRT-PCR) confirmed a high expression level of hsa-miR-223 in macrophages but not in SKBR3 or MDA-MB-231 cells. * p < 0.05. (C) Co-culture of breast cancer cells with macrophages increased miR-223 levels in breast cancer cells. Breast cancer cell lines (SKBR3 and MDA-MB-231) were cultured alone (blank) or co-cultured with unactivated or IL-4-activated macrophages without direct cell-cell contact. miR-223 expression in SKBR3 and MDA-MB-231 cells was detected using qRT-PCR. Relative levels of miR-223 expression normalised to U6 rRNA levels are presented. * p < 0.05. (D) Functional assay of miR-223 in breast cancer cells. miR-223-targeting luciferase reporter containing a miR-223 complementary sequence within the 3'-UTR of the luciferase reporter gene was constructed. SKBR3 cells were transfected with the reporter gene and cultured alone (blank) or co-cultured with IL-4-activated macrophages. As controls, SKBR3 cells were co-transfected with the reporter gene and miR-NC, miR-660 or miR-223. Relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p < 0.05.