MiR-214-3p Regulates Apoptosis of Ameloblasts under Excessive Fluoride via PI3K/AKT Signaling Pathway

Abstract

Background: Excessive intake of fluoride during enamel growth and development can impair the normal physiological function of ameloblasts, resulting in the formation of dental fluorosis. However, little is known about the function of miRNAs in the formation of dental fluorosis. Aim: This study aimed to explore the effects of key miRNAs on the PI3K/AKT signaling pathway and ameloblasts under high fluoride conditions. Materials and Methods: LS8 cells were treated with NaF at concentrations of 0.4, 0.8, 1.6, 3.2, and 6.4 mmol/L for 24 h, and cell viability and apoptosis were measured using the CCK-8 assay and flow cytometry. The expression of apoptosis-related proteins was detected by Western blotting. Transcriptome sequencing was performed on FS8 cells after treatment with 1.6 and 3.2 mmol/L NaF for 24 h to identify key miRNAs and validate them. After cell transfection, the effect of miR-214-3p on ameloblasts and the PI3K/AKT signaling pathway was assessed. Results and Discussion: NaF treatment significantly reduced the viability and accelerated the apoptosis of LS8 cells. The down-regulated miRNAs predicted target genes that were most enriched in the PI3K/AKT signaling pathway, and the most critical miRNA was miR-214-3p. The expression levels of p-PI3K, p-AKT, and Bcl-2 were significantly up-regulated after overexpression of miR-214-3p in LS8 cells, while the expression of PI3K, AKT, and Bax was significantly down-regulated, which was partially reversed by LY294002. Conclusion: Excess fluoride could affect the morphology of ameloblast-like cell lines and induce apoptosis. Overexpression of miR-214-3p inhibited NaF-induced apoptosis in LS8 cells by regulating the PI3K/AKT signaling pathway, inhibiting its phosphorylation, down-regulating the Bax protein, and up-regulating the Bcl-2 protein.