Background

Endemic fluorosis remains a major public health issue in many countries. Fluoride can cause abnormalities in osteoblast proliferation and activation, leading to skeletal fluorosis. However, its detailed molecular mechanism remains unclear. Based on a previous study, the aim of this study is to explore the role of miRNA in osteoblast activation of skeletal fluorosis via targeting of Cyclin D1.

Methods

A population study of coal-burning fluorosis and in vitro experiments were performed in this study. Urine fluoride (UF) concentrations of the participants were determined using a national standardized ion selective electrode approach. Based on our previous miRNA sequence results, bioinformatic analysis was used to predict miR-4755-5p targeting Cyclin D1. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of miR-4755-5p. The expression of Cyclin D1 mRNA was detected by qRT-PCR. The expression of Cyclin D1 protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. Cell viability was detected by CCK-8 method. The distribution of the cell cycle was analyzed by flow cytometry. The alkaline phosphatase (ALP) activity and bone Gla protein (BGP) content were detected by micronutrient enzymes standard method and ELISA. The target binding between miR-4755-5p and Cyclin D1 was verified using dual-luciferase reporter assay.

Results

In the fluoride-exposed population, the results showed that with the increase in UF content, the expression of miR-4755-5p decreased gradually, while the mRNA transcription and protein expression of Cyclin D1 increased gradually. The relative miR-4755-5p expression showed a negative correlation with Cyclin D1 expression. Subsequently, in human osteoblasts treated with sodium fluoride (NaF), the results also showed that NaF caused low expression of miR-4755-5p and increased expression of Cyclin D1. Further, the results of miR-4755-5p mimic transfection confirmed that under the action of NaF, miR-4755-5p overexpression reduced Cyclin D1 protein expression within osteoblasts and further inhibited cell proliferation and activation. Simultaneously, luciferase reporter assays verified that Cyclin D1 was the miR-4755-5p direct target.

Conclusion

The results demonstrate that fluoride exposure induced the downregulation of miR-4755-5p and downregulated miR-4755-5p promoted fluoride-induced osteoblast activation by increasing Cyclin D1 protein expression. This study sheds new light on biomarkers and potential treatment for endemic fluorosis.

中文翻译：

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miR-4755-5p 的下调通过标记 Cyclin D1 促进氟化物诱导的成骨细胞活化。

背景

地方性氟中毒仍然是许多国家的一个主要公共卫生问题。氟化物会导致成骨细胞增殖和活化异常，导致氟骨症。然而，其详细的分子机制仍不清楚。基于之前的研究，本研究的目的是探讨miRNA通过靶向Cyclin D1在氟骨症成骨细胞激活中的作用。

方法

本研究进行了燃煤氟中毒的人群研究和体外实验。使用国家标准化离子选择电极方法测定参与者的尿液氟化物（UF）浓度。根据我们之前的 miRNA 序列结果，使用生物信息学分析来预测 miR-4755-5p 靶向Cyclin D1。使用实时定量PCR（qRT-PCR）来验证miR-4755-5p的表达。qRT-PCR检测Cyclin D1 mRNA的表达。分别采用酶联免疫吸附试验(ELISA)和Western blotting检测Cyclin D1蛋白的表达。采用CCK-8法检测细胞活力。通过流式细胞术分析细胞周期的分布。采用微量营养素酶标准法和ELISA法检测碱性磷酸酶（ALP）活性和骨Gla蛋白（BGP）含量。使用双荧光素酶报告基因测定验证miR-4755-5p 和Cyclin D1之间的靶结合。

结果

在氟化物暴露人群中，结果显示，随着UF含量的增加，miR-4755-5p的表达量逐渐下降，而Cyclin D1的mRNA转录和蛋白表达量逐渐增加。miR-4755-5p的相对表达量与Cyclin D1表达量呈负相关。随后，在用氟化钠（NaF）处理的人成骨细胞中，结果还表明NaF导致miR-4755-5p的低表达和Cyclin D1的表达增加。进一步，miR-4755-5p模拟转染的结果证实，在NaF的作用下，miR-4755-5p过表达降低了成骨细胞内Cyclin D1蛋白的表达，并进一步抑制细胞增殖和活化。同时，荧光素酶报告基因检测证实Cyclin D1是 miR-4755-5p 的直接靶标。

结论

结果表明，氟化物暴露诱导 miR-4755-5p 下调，并且下调 miR-4755-5p 通过增加 Cyclin D1 蛋白表达来促进氟化物诱导的成骨细胞活化。这项研究为地方性氟中毒的生物标志物和潜在治疗提供了新的线索。