Fig. 1.

MiR-222 is upregulated in breast cancer-associated fibroblasts (CAFs), and controls breast fibroblast phenotype. a MiR-222 expression was determined by qPCR, and is shown for CAFs relative to NFs in three separate cell types. Left plot: matched pairs of normal fibroblasts (NFs) and CAFs were isolated from breast cancer patient samples using laser microdissection of archival (fixed) tissue. The data represent technical triplicates. Middle plot: four matched pairs of primary cultured CAFs and NFs from breast cancer patient-derived tumour samples. The data represent technical triplicates. Right plot: immortalised breast CAF and NF cell lines. The data represent biological triplicates. b Immortalised breast NFs (left) or CAFs (right) were transfected with miR-222 mimics or control (NC), or miR-222 inhibitor (i) or control (NC) and miR-222 expression was assessed using qPCR. Data represent biological triplicates. c Relative gene expression levels of the CAF markers, α-SMA, Fibroblast Specific Protein (FSP), CCL2 and VEGF were assessed in immortalised CAFs as compared with immortalised NFs using qPCR. The data represent biological triplicates. d Relative expression levels of the same CAF markers were assessed using qPCR in NFs (left) or CAFs (right) transfected with miR-222 mimics or control (NC), or miR-222 inhibitor (i) or control (NC). The data represent biological triplicates. e NFs or CAFs were transfected with miR-222 mimics or control (NC), or miR-222 inhibitor (i) or control (NC) and protein expression of the CAF markers α-SMA (ACTA2) and vimentin were assessed using Western blots, along with β-actin as a loading control. ***p < 0.0005 and **p < 0.005