Fig. 5: NGF down-regulates the expression of miR-92a-1-5p through the MEK/ERK signaling pathway, increases the expression of MMP-2 and promotes the migration of osteosarcoma cells.

A An analysis of 2 miRNA prediction databases predicted 25 miRNAs that bind with MMP-2 3’-UTR. B, C The osteosarcoma cells were treated with NGF and miRNAs were screened using a qPCR assay. D, E Osteosarcoma cells were treated with different concentration of NGF (30, 50, 100 ng/mL) for 24 h and miRNAs expression were examined by qPCR. F–N Osteosarcoma cells were transfected with miR-92a-1-5p mimic or control mimic for 24 h, then stimulated with NGF for 24 h. The levels of MMP-2 and cell migration were examined by qPCR, Western blot, wound healing, and the Transwell migration (scale bar 500 μm). O The MMP-2 3’-UTR wild-type and mutant contained the miR-92a-1-5p binding site. P Osteosarcoma cells were transfected with the MMP-2 3’-UTR wild-type or mutant plasmid for 24 h, then stimulated with NGF for 24 h, and relative luciferase activity was measured. Q Osteosarcoma cells were co-transfected with miR-92a-1-5p mimic and MMP-2 3’-UTR wild-type for 24 h, then stimulated with NGF for 24 h, and relative luciferase activity was measured. R, S Osteosarcoma cells were pretreated with MEK and ERK inhibitors for 30 min, or transfected siRNA for 24 h, then stimulated with NGF for 24 h, and the miR-92a-1-5p expression was quantified by qPCR. All experiments were repeated 3 to 5 times. *p < 0.05 compared with the control group; ^#p < 0.05 compared with the NGF-treated group.