2022, Vol. 26 ›› Issue (5): 682-687

Overexpression of miR-25 downregulates titanium particle-induced osteoclast differentiation through the NFATc1 signaling pathway

Hu Weifan1, Zheng Li1, Li Dadi1, Sun Yang1, Zhao Fengchao2   

Abstract: BACKGROUND: Studies have found that the down-regulation of miR-25 is related to the excessive activation of nuclear factor of activated T cells c1 (NFATc1), and NFATc1 is the core transcription factor in osteoclasts differentiation and maturation, suggesting that the expression level of miR-25 can regulate osteoclasts differentiation.
OBJECTIVE: To explore the effect of miR-25 overexpression on titanium (Ti) particles-induced osteoclasts differentiation and investigate the signaling pathways.
METHODS: RAW264.7 cell line was cultured in vitro. Cell counting kit-8 was used to determine the effect of Ti particles on cell proliferation. miR-25 mimics were transfected into RAW264.7 cells, and cultured with Ti particles to induce osteoclasts differentiation. Immunofluorescence was used to detect the activation status of NFATc1, TRAP staining and counting were adopted to verify the differentiation of osteoclasts, and real-time PCR was employed to detect and analyze the differential expression of NFATc1, CaMKII, and CaMKIV mRNAs.
RESULTS AND CONCLUSION: The low concentration of Ti particles (0.1 g/L) had no significant effect on cell proliferation. Compared with the negative control group, there was less production of osteoclasts (TRAP positive) in the miR-25 overexpression group (P < 0.05). After intracellular transfection with miR-25 mimic, immunofluorescence detection showed that the activation of NFATc1 decreased and the fluorescence intensity decreased. The expression of signaling pathway molecules NFATc1, CaMKII, and CaMKIV mRNAs decreased significantly after miR-25 overexpression (P < 0.05). These findings indicate that miR-25 overexpression can inhibit the differentiation of Ti particles-induced osteoclasts. This process may be achieved through the Ca2+/NFATc1 pathway.

Key words:miR-25, osteoclast, NFATc1, RAW264.7 cell, titanium particle