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. 2016 Oct 12;7(48):79032–79046. doi: 10.18632/oncotarget.12593

Figure 5. Inhibition of miR-218-5p in breast cancer cells decreases PTHrP expression, reduces Rankl in osteoblasts and impairs osteoclast differentiation.

Figure 5

A. PTHrP expression in bone metastases was determined by immunohistochemistry. Scale bar indicates 100 μm (upper panel) and 50 μm (lower panel). B. PTHrP mRNA expression was analyzed in MDA-MB-231 cells delivered with miR-218-5p upon inhibition of Wnt signaling using a small molecule inhibitor CCT036477. C. Cells were transfected with antimiR-218-5p and Wnt signaling was activated by Lithium Chloride. PTHrP mRNA expression was determined by qRT-PCR. D. Conditioned medium (CM) was collected from MDA-MB-231 cells transfected with miR-218-5p mimic or inhibitor and PTHrP protein level was determined using ELISA. E. Long bone osteoblasts were incubated with CM collected from miR-218-5p mimic or inhibitor -delivered MDA-MB-231 cells. Rankl expression was determined in osteoblasts by qRT-PCR. F. Osteoblast-osteoclast co-cultures were performed in the presence of CM from MDA-MB-231 cells transfected with miR-218-5p or antimiR-218-5p. G. Osteoclasts were stained with TRAP and TRAP-positive multinucleated cells (MNCs) were counted. H. Model of miR-218 function in the metastatic bone environment. Minimum of 3 independent experiments. Mean values ± SEM, *p<0.05, ** p < 0.01 vs. miR-Ctrl.