let-7c-5p regulates CyclinD1 in fluoride-mediated osteoblast proliferation and activation

Abstract Endemic fluorosis is caused by the intake of high environmental fluoride levels, which causes dental and skeletal fluorosis. Osteoblast proliferation and activation is closely related to skeletal fluorosis and is tightly regulated by the cell cycle. Several biological processes, including bone metabolism and osteoblast proliferation and activation, are regulated by a type of non-coding RNA called microRNAs (miRNAs). However, the understanding of miRNA functions in skeletal fluorosis is limited. Based on our previous miRNA sequencing results and bioinformatics analysis, we investigated the function of the miRNA let-7c-5p to regulate CyclinD1 in fluoride-induced osteoblast proliferation and activation. We designed population experiments as well as in vitro studies using EdU, flow cytometry, immunofluorescence, dual-luciferase reporters, and chromatin immunoprecipitation. The population-based analysis showed a decrease in let-7c-5p expression as fluoride exposure increased. In addition, let-7c-5p levels were negatively correlated with CyclinD1 and Wnt9a (another let-7c-5p target). We verified in vitro that let-7c-5p participates in the fluoride-induced proliferation and activation of human osteoblasts by directly targeting CyclinD1. Furthermore, we demonstrated that let-7c-5p regulates CyclinD1 expression via the Wnt/β-catenin signaling pathway. This study demonstrated the participation of let-7c-5p in fluoride-induced proliferation and activation of human osteoblasts by regulation of CyclinD1 expression at the post-transcriptional and transcriptional levels. We report for the first time that the Wnt9a/β-catenin/CyclinD1 axis is involved in fluorosis development.