Effects of fluoride on the proliferation and activation of osteoblasts by regulating methylation of the DNA repair genes MGMT and MLH1

Long Chen; Na Yin; Yi Ding; Mei-Lin Zhang; Min Li; Jin-Jie Zhong; Shu-Mei Feng

doi:10.1016/j.reth.2022.01.004

Effects of fluoride on the proliferation and activation of osteoblasts by regulating methylation of the DNA repair genes MGMT and MLH1

Regen Ther. 2022 Jan 30:19:107-112. doi: 10.1016/j.reth.2022.01.004. eCollection 2022 Mar.

Authors

Long Chen¹, Na Yin¹, Yi Ding², Mei-Lin Zhang³, Min Li², Jin-Jie Zhong⁴, Shu-Mei Feng²

Affiliations

¹ Functional Center, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, Xinjiang, 830011, China.
² Department of Histology and Embryology, School of Basic Medical Sciences, Xinjiang Medical University, Urumqi, Xinjiang, 830011, China.
³ Cilinical Laboratoray, Urumqi Blood Center, Urumqi, Xinjiang, 830000, China.
⁴ Department of Basic Medicine Sciences, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, China.

Abstract

Introduction: Fluoride can induce the proliferation and activation of osteoblasts, resulting in skeletal fluorosis progression; however, the specific mechanism is unclear.

Methods: Cell proliferation was examined using the MTT assay. Flow cytometry was performed to detect the cell cycle distribution. Alkaline phosphatase (ALP) was calculated to evaluate bone formation and turnover. Gene methylation was examined using the MSP assay. mRNA and protein expression levels were assessed using qRT-PCR and Western blot assays.

Results: Low-concentration NaF treatment promoted the cell cycle progression of osteoblasts to S-phase, thus accelerating cell proliferation and activation in a concentration-dependent manner. In addition, the methylation of the MGMT and MLH1 genes was increased, and their mRNA expression was reduced. Furthermore, the DNA methyltransferase inhibitor 5-AZA-dC suppressed cell viability, cell number in S-phase, ALP activity and osteogenesis-related protein levels in osteoblasts treated with low doses of NaF. Meanwhile, 5-AZA-dC suppressed the increase in MGMT and MLH1 gene methylation in osteoblasts treated with low doses of NaF, leading to enhanced expression of MGMT and MLH1 mRNA.

Conclusion: NaF treatment led to methylation of the DNA repair genes MGMT and MLH1 in osteoblasts, resulting in cell proliferation and activation and causing the development of skeletal fluorosis.

Keywords: 5-AZA-dC, 5-aza-2-deoxycytidine; ALP, Alkaline phosphatase; BGP, bone gla protein; BLBC, Basal-like breast cancer; BMP, Bone morphogenetic protein; MGMT; MGMT, O6-Methylguanine-DNA methyltransferase; MLH1; MSP, Methylation specific PCR; Methylation; NaF; NaF, Sodium fluoride; Osteoblasts; Skeletal fluorosis.