Abstract: BACKGROUND: Fluorosis is a disorder of enamel development caused by long-term intake of large amounts of fluoride during enamel development.
OBJECTIVE: To further explore the molecular mechanism of dental fluorosis formation by screening the differentially expressed genes associated with calcium homeostasis in ameloblasts by transcriptome sequencing technology.
METHODS: LS8 cells were treated with 0, 0.4, 0.8, 1.6, 3.2 and 6.4 mmol/L sodium fluoride (NaF) for 24, 48 and 72 hours to observe the effects of different concentrations of NaF on the morphology, cell activity and intracellular Ca2+ concentration of LS8 cells. The differentially expressed genes were screened by transcriptome sequencing and validated.
RESULTS AND CONCLUSION: After 24 hours of treatment, the cells treated with 0, 0.4, and 0.8 mmol/L NaF were in good growth condition, with increased cell number and clear cell outline. When the NaF concentration was ≥ 1.6 mmol/L, the cells were gradually shrunken and became smaller and the number of cells decreased with the increase of NaF concentration. After 48 and 72 hours of treatment, the number of cells increased in the 0, 0.4 mmol/L NaF groups, while gradually decreased in the 0.8, 1.6, 3.2 mmol/L NaF groups, with rounded and smaller cell morphology. The cells in the 6.4 mmol/L NaF group were shrunken, rounded and suspended in the medium, with almost no adherent cells. When treated with the same concentration of NaF, LS8 cells were in optimal growth after 24 hours of treatment. Results from cell counting kit-8 assay showed that when treated with the same concentration of NaF, the cell activity decreased with the increase of treatment time; when the treatment time was the same, the cell activity decreased with the increase of NaF concentration. After 24 hours of treatment, the intracellular Ca2+ concentration increased with the increase of NaF concentration. Transcriptome sequencing analysis identified genes involved in the regulation of cellular calcium homeostasis: Hsp90b1, Canx, Calr, and Hspa5 that were significantly upregulated (P < 0.05) and Cacna1a that was significantly downregulated (P < 0.05). To conclude, the inhibitory effect of NaF on LS8 cell proliferation may be related to the abnormal increase in intracellular Ca2+ concentration, and the mechanism may be caused by the upregulation of the expression of protein processing and synthesis pathways Hsp90b1, Canx, Calr, and Hspa5 and the downregulation of the expression of calcium signaling pathway Cacna1a.

Key words: fluorosis, dental fluorosis, ameloblast, sodium fluoride, transcriptome sequencing, endoplasmic reticulum stress, calcium channel, calcium probe