The effects of fluoride on hypermethylation, transcription and expression of p16 gene in osteoblasts of rats

Abstract

Objective To establish an in vitro model of primary osteoblasts fluorosis and to detect the influences of different doses of fluorosis on promoter methylation, transcription and expression of p16, then study the epigenetic effects of p16 gene on skeletal fluorosis. Methods Osteoblasts were isolated from Sprague- Dawley neonatal rats by enzyme digestion, and identified by morphology, alkaline phosphatase staining and alizarin red staining. Osteoblast were treated with 0 (control group), 200, 400, 800 and 1 600 μmol/L NaF for 72 h. The p16 gene promoter region was amplified in the transcription initiation site - 88 - + 143 region by bisulfate-sequencing polymerase chain reaction (BSP). The mRNA transcription and the protein expression of p16 were detected by realtime quantitative PCR and Western blotting. Results Among the groups of osteoblasts treated with 200, 400, 800, 1 600 μmol/L NaF, the positive rates of DNA methylation of promoter region in p16 gene of osteoblasts were 5.88% (10/170), 12.94% (22/170), 17.65% (30/170) and 33.53% (57/170), respectively. No DNA methylation was observed in the control group. There were significant differences between the control group and the NaF- treated osteoblasts groups (χ2 = 92.87,P < 0.05). Average levels of p16 mRNA were 1.050 ± 0.073, 0.869 ± 0.037, 1.065 ± 0.118 and 0.786 ± 0.148 in 200, 400, 800 and 1 600 μmol/L NaF-treated osteoblasts groups, compared with the control group(1.110 ± 0.315), there were significant differences among groups (allP < 0.05) and 1 600 μmol/L NaF- treated osteoblasts group was much lower than other groups (P < 0.05). Average levels of p16 protein were 1.190 ± 0.050, 1.214 ± 0.058, 1.122 ± 0.123 and 0.320 ± 0.074 in 200, 400, 800 and 1 600 μmol/L NaF-treated osteoblasts groups, compared with the control group (1.115 ± 0.057), there were significant differences among groups (allP < 0.05) and 1 600 μmol/L group was much lower than other groups. Conclusion NaF can cause hypermethylation in the promoter region of p16 gene, then suppress the expression of mRNA and protein, which might be one of the important mechanisms of cell proliferation change and cell cycle disorder in skeletal fluorosis. Key words: Fluoride; Osteoblast; p16 gene; DNA methylation