Downregulation of miR-4755-5p promotes fluoride-induced osteoblast activation via tageting Cyclin D1
Downregulation of miR-4755-5p promotes fluoride-induced osteoblast activation via tageting Cyclin D1
复制标题
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。
DOI：
10.1016/j.jtemb.2020.126626
复制
发表时间：
2020-12-01
影响因子：
3.5
通讯作者：
Pan, Xueli
中科院分区：
文献类型：
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作者：
Gao, Jiayu;Qin, Yu;Pan, Xueli
背景：地方性氟中毒在许多国家仍然是一个主要的公共卫生问题。氟可引起成骨细胞增殖和激活的异常，导致氟骨症。然而，其详细的分子机制仍不清楚。本研究的目的是在前人研究的基础上，通过靶向Cyclin D1，探讨miRNA在氟骨症成骨细胞活化中的作用。采用国家标准的离子选择电极法测定受试者的尿氟浓度。基于我们以前的miRNA序列结果，利用生物信息学分析预测了靶向Cyclin D1的miR-4755-5p。用实时定量聚合酶链式反应(qRT-PCR)检测miR-4755-5p的表达。采用定量逆转录聚合酶链式反应(qRT-PCR)检测细胞周期蛋白D1mRNA的表达。分别用酶联免疫吸附试验(ELISA)和Western blotting检测Cyclin D1蛋白的表达。CCK-8法检测细胞存活率。流式细胞仪分析细胞周期分布。用微量营养酶标准法和酶联免疫吸附试验检测碱性磷酸酶(ALP)活性和骨钙素(BGP)含量。结果：在氟暴露人群中，随着尿氟含量的增加，miR-4755-5P的表达逐渐降低，而Cyclin D1的mRNA转录和蛋白表达逐渐增加。MiR-4755-5p的相对表达与Cyclin D1的表达呈负相关。随后，在氟化钠(NaF)处理的人成骨细胞中，结果也显示NaF导致miR-4755-5p的低表达和Cyclin DL的表达增加。此外，miR4755-5p模拟转染结果证实，在NaF作用下，miR-4755-5p过表达降低了成骨细胞内Cyclin D1蛋白的表达，进一步抑制了细胞的增殖和激活。结论：氟暴露诱导miR-4755-5p表达下调，miR-4755-5p表达下调，且miR-4755-5p表达下调通过上调Cyclin D1蛋白表达促进成骨细胞活化。这项研究为地方性氟中毒的生物标志物和潜在的治疗方法提供了新的线索。
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 Dl. Further, the results of miR4755-5p 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.