The effect of melatonin on the mouse ameloblast-lineage cell line ALCs

Abstract

Melatonin plays a critical role in promoting the proliferation of osteoblasts and the growth and development of dental papilla cells. However, the effect and mechanism of melatonin on the growth and development of ALCs still need to be explored. CCK8 assay was used for the evaluation of cell numbers. qRT-PCR was used to identify the differentially expressed genes in ALCs after melatonin treatment. The number and morphology of ALCs were investigated by confocal microscopy. Alkaline phosphatase assay and Alizarin red S staining were used for measuring mineralization. Then, we focused on observing the crucial factors of the signaling pathway by RNA-seq and qRT-PCR. Melatonin limited the cell number of ALCs in a dose-dependent manner and promoted the production of actin fibers. A high concentration of melatonin significantly promoted the mRNA levels of enamel matrix proteins and the formation of mineralized nodules. RNA-seq data showed that Wnt signaling pathway may be involved in the differentiation of ALCs under the influence of melatonin. This study suggests that melatonin plays a regulatory role in the cell number, differentiation, and mineralization of the ALCs, and then shows the relationship between the Wnt signaling pathway with the ALCs under melatonin.

Figure 1

Effect of melatonin on ALCs’ cell number. Cells were treated with various concentrations of MT (0, 10^–10, 10^–8, 10^–6, 10^–4 and 10⁻³ M) for 24, 48, 72 and 96 h respectively. Subsequently, the viability of the cells was measured via a CCK8 assay. Each value was presented as mean ± SD for triplicate cultures. (A) Growth curve of ALCs in different concentrations of MT at different periods. (B) The proliferation rate of ALCs in different concentrations of MT at different periods. (*p < 0.05, **P < 0.001, compared to control).

Figure 2

Effect of melatonin on ALCs’ morphology. Cells were treated with various MT concentrations (0, 10^–10, 10^–8, 10^–6, 10^–4, and10⁻³ M) for 48 and 96 h, respectively. Subsequently, the cells were observed by laser scanning confocal microscope (LSCM). The magnification is 50 μm. Cells were treated with MT concentrations for 48 h (A) and 96 h (B), respectively.

Figure 3

Effect of melatonin on expression of specific enamel genes in ALCs. Cells were treated with various concentrations of MT (0, 10^–4 and10⁻³ M) for 1, 2, 3, 4, and 5d. Subsequently, the expression of specific enamel genes and MTNRs was measured in qRT-PCR. (*p < 0.05, **P < 0.001, compared to control).

Figure 4

Effect of melatonin on ALCs’ mineralization. (A) ALP activity in ALCs under MT treatments (0, 1 × 10^–4, 2 × 10^–4, 5 × 10^–4, 1 × 10⁻³ M) was determined on day 7. Each value was presented as mean ± SD for triplicate experiments. (*p < 0.05, **P < 0.001, compared to control). The magnification is 200 μm. (B) Effect of melatonin on the formation of the mineralized nodule in ALCs. Cells were treated with various concentrations of MT (0, 1 × 10^–4, 2 × 10^–4, 5 × 10^–4, 1 × 10⁻³ M) for 28 days. (C) Effect of melatonin and Luzindole on the formation of mineralized nodules of ALCs. Cells were incubated with the conditioned medium ((L-MT−), (L + MT−), (L-MT+), (L + MT+)) for 28 days.

Figure 5

RNA sequencing Analysis. MT: melatonin, CTRL: control. (A) Venn diagram revealed unique and overlapping gene expression between CTRL and MEL. (B) Volcano plots revealed significant changes in [MT (n = 2) relative to CTRL (n = 2)] genes in ALCs. Each dot in the volcano plots represents a detected gene. In the MT group, the genes with significantly increased [fold change > 2, P < 0.001] were shown in red. While those in CTRL group significantly decreased [fold change < 2, P < 0.001] were shown in blue. (C) KEGG enrichment analysis of DEGs in ALCs (Only the top 20 KEGG signaling pathways were selectively extracted). The vertical axis indicates the enrichment of KEGG signaling pathways. (D) The heat map of Wnt signaling pathway-related gene expression in the control group and melatonin treated ALCs for 3d. (E) The main factors of the Wnt canonical pathway in ALCs treated with/without melatonin for 3d were verified by qRT-PCR.