Fluoride is a persistent environmental pollutant, and its excessive intake contributes to skeletal and dental fluorosis. The mechanisms underlying fluoride-induced abnormal osteoblast proliferation and activation, which are related to skeletal fluorosis, have not yet been fully clarified. As important epigenetic regulators, microRNAs (miRNAs) participate in bone metabolism. On the basis of our previous miRNA-seq results and bioinformatics analysis, this study investigated the role and specific molecular mechanism of miR-486-3p in fluoride-induced osteoblast proliferation and activation via CyclinD1. Herein, in the fluoride-challenged population, we observed that miR-486-3p expression decreased while CyclinD1 and transforming growth factor (TGF)-?1 increased, and miR-486-3p level correlated negatively with the expression of CyclinD1 and TGF-?1 genes. Further, we verified that sodium fluoride (NaF) decreases miR-486-3p expression in human osteoblasts and overexpression of miR-486-3p reduces fluoride-induced osteoblast proliferation and activation. Meanwhile, we demonstrated that miR-486-3p regulates NaF-induced upregulation of CyclinD1 by directly targeting its 3?-untranslated region (3?-UTR). In addition, we observed that NaF activates the TGF-?1/Smad2/3/CyclinD1 axis and miR-486-3p mediates transcriptional regulation of CyclinD1 by TGF-?1/Smad2/3 signaling pathway via targeting TGF-?1 3?-UTR in vitro. This study, thus, contributes significantly in revealing the mechanism of miR-486-3p-mediated CyclinD1 upregulation in skeletal fluorosis and sheds new light on endemic fluorosis treatment.
*Original abstract online at https://onlinelibrary.wiley.com/doi/10.1002/tox.23302
Supplementary Table 1 Primers of mRNA used in qRT-PCR.
5?- CTGTACATTGACTTCCGCAAG -3?
|GAPDH F||5?-GCACCGTCAAGGCTGAGAAC -3?|
|GAPDH R||5?- TGGTGAAGACGCCAGTGGA -3?|
Supplementary Table 2 Primers used in ChIP-qRT-PCR.
|ChIP 1 F||5?- ACACCCCCAACAAAACCAA -3?|
|ChIP 1 R
ChIP 2 F
ChIP 2 R
5?- TGCCAACCTCCTCAACGAC -3?
5?- AGGGGGGTGAGTAGCAAAGA -3?
Funding information: National Natural Science Foundation of China, Grant/Award Number: 81660524; The First-Class Discipline Construction Project in Guizhou Province-Public Health and Preventive Medicine, Grant/Award Number: 2017