Fig. 4: miR-182 upregulated NSCLC cells promote osteoclastogenesis through enhancing IL-8 secretion.

A TRAP staining analysis of the osteoclast differentiation of RAW264.7 cells after cultured in the indicated CM for 6 days. Arrowheads indicate mature, multi-nucleated osteoclasts. B Transcriptome-seq combined with Gene Ontology analysis revealed the enrichment of multiple signaling pathways in miR-182-overexpressing A549 cells. C Volcano plot showing differentially expressed genes in miR-182-overexpressing A549 cells compared with the negative control cells, with selected cytokines highlighted. D qPCR analysis of the mRNA expression of IL-8, IL-1, IL-12 and CXCL3 in miR-182-overexpressing A549 (left) or H1299 cells relative to controls (right). E, F Western blotting of IL-8, IL-1, IL-12, and CXCL3 levels in CM from miR-182-overexpressing (left) or H1299 cells (right) relative to controls, with total protein staining (Coomassie) confirmed equal loading. F, G ELISA analysis of IL-8, IL-1, IL-12, and CXCL3 levels in the CM from miR-182-overexpressing (top) or H1299 cells (bottom) relative to controls (F) or in blood samples collected from mice bearing bone metastases developed by miR-182-overexpressing A549 cells or NC A549 cells (G). (H) TRAP staining analysis of the effects of depletion of indicated factors in the miR-182-CM (top) or their addition to the CM from A549 cells (bottom) on osteoclast differentiation of RAW264.7 cells. Arrowheads indicate mature, multi-nucleated osteoclasts. Scale bars, 50 μm. The average values ± SEM of three separate experiments are plotted. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.