Fluorine is widely dispersed in nature and has multiple physiological functions. Although it is usually regarded as an essential trace element for humans, this view is not held universally. Moreover, chronic fluorosis, mainly characterized by skeletal fluorosis, can be induced by long-term excessive fluoride consumption. High concentrations of fluoride in the environment and drinking water are major causes, and patients with skeletal fluorosis mainly present with symptoms of osteosclerosis, osteochondrosis, osteoporosis, and degenerative changes in joint cartilage. Etiologies for skeletal fluorosis have been established, but the specific pathogenesis is inconclusive. Currently, active osteogenesis and accelerated bone turnover are considered critical processes in the progression of skeletal fluorosis. In recent years, researchers have conducted extensive studies in fields of signaling pathways (Wnt/β-catenin, Notch, PI3K/Akt/mTOR, Hedgehog, parathyroid hormone, and insulin signaling pathways), stress pathways (oxidative stress and endoplasmic reticulum stress pathways), epigenetics (DNA methylation and non-coding RNAs), and their inter-regulation involved in the pathogenesis of skeletal fluorosis. In this review, we summarised and analyzed relevant findings to provide a basis for comprehensive understandings of the pathogenesis of skeletal fluorosis and hopefully propose more effective prevention and therapeutic strategies.

Progress of Signaling Pathways, Stress Pathways and Epigenetics in the Pathogenesis of Skeletal Fluorosis.

Excessive intake of fluoride may cause female reproductive dysfunction but pathological mechanism is unclear. The miRNAs in follicular fluid are a class of small non-coding RNAs from granulosa cells. The aim of this study is to examine the differential expressions of miRNAs in ovarian granulosa cells from women suffering from fluorosis and infertility. Five fluorosis women suffering infertility and 5 non-fluorosis (normal ovarian secretory function control) women were included as two groups. These two groups were indexed by serum and urine fluoride (F) levels as high F group and control group. The concentrations of estradiol (E2), progesterone (P), human chorionic gonadotropin (HCG), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were measured by ELISA kits. The characteristics of menstruation from each woman were collected. The total RNA was isolated from granulosa cells for sequencing. The validation was completed by RT-qPCR. There was no significant difference between the two groups on age, the levels of E2, FSH, P, HCG, and LH in serum, and the characteristics of menstruation. A total of 37 miRNAs were upregulated and 40 miRNAs downregulated in follicular fluid from granulosa cells in the high F group compared with that in the control group. The expression of hsa-miR-29b increased in fluorosis patients identified by the miRNA sequence analysis. The distinct expression patterns of miRNAs in women requiring IVF from fluorosis area provide a new direction for molecular mechanism of fluorosis caused reproductive dysfunction.

Analysis of the MicroRNA Profile by Sequencing in Ovarian Granular Cells from Women Suffering Fluorosis with Reproductive Dysfunction

Chronic intake of fluoride, existing in the environment, may cause endemic fluorosis, which is characterized by the occurrence of skeletal and dental fluorosis. However, the pathogenesis of fluorosis has not yet been elucidated. Abnormal osteoblast proliferation and activation have a pivotal role in bone turnover disorders which are linked to skeletal fluorosis. MicroRNAs are involved in fundamental cellular processes, including cell proliferation. Based on our previous study, population study and in vitro experiments were designed to understand the effect of miR-122-5p on osteoblast activation in skeletal fluorosis through targeting cyclin-dependent kinase 4 (CDK4). In human populations with coal-burning type fluoride exposure, the results showed that miR-122-5p was downregulated but CDK4 expression was upregulated and miR-122-5p was negatively correlated with CDK4 expression. Furthermore, in human osteoblasts treated with sodium fluoride, we demonstrated that miR-122-5p mediated osteoblast activation of skeletal fluorosis via upregulation of the CDK4 protein. In support of this, dual-luciferase reporter assay showed that miR-122-5p modulated CDK4 protein levels by targeting its 3′-untranslated region. These findings show, for the first time, that miR-122-5p may be involved in the cause and development of skeletal fluorosis by targeting CDK4.

miR-122-5p Mediates Fluoride-Induced Osteoblast Activation by Targeting CDK4

Let-7c-5p Regulates CyclinD1 in Fluoride-Mediated Osteoblast Proliferation and Activation.

Fluoride is a persistent environmental pollutant, and its excessive intake contributes to skeletal and dental fluorosis The mechanisms underlying fluoride-induced abnormal osteoblast proliferation and activation, which are related to skeletal fluorosis, have not yet been fully clarified As important epigenetic regulators, microRNAs (miRNAs) participate in bone metabolism On the basis of our previous miRNA-seq results and bioinformatics analysis, this study investigated the role and specific molecular mechanism of miR-486-3p in fluoride-induced osteoblast proliferation and activation via CyclinD1 Herein, in the fluoride-challenged population, we observed that miR-486-3p expression decreased while CyclinD1 and transforming growth factor (TGF)-β1 increased, and miR-486-3p level correlated negatively with the expression of CyclinD1 and TGF-β1 genes Further, we verified that sodium fluoride (NaF) decreases miR-486-3p expression in human osteoblasts and overexpression of miR-486-3p reduces fluoride-induced osteoblast proliferation and activation Meanwhile, we demonstrated that miR-486-3p regulates NaF-induced upregulation of CyclinD1 by directly targeting its 3'-untranslated region (3'-UTR) In addition, we observed that NaF activates the TGF-β1/Smad2/3/CyclinD1 axis and miR-486-3p mediates transcriptional regulation of CyclinD1 by TGF-β1/Smad2/3 signaling pathway via targeting TGF-β1 3'-UTR in vitro This study, thus, contributes significantly in revealing the mechanism of miR-486-3p-mediated CyclinD1 upregulation in skeletal fluorosis and sheds new light on endemic fluorosis treatment

miR-486-3p regulates CyclinD1 and promotes fluoride-induced osteoblast proliferation and activation.

MicroRNAs (miRNAs) differentially expressed in plasma were identified using microRNA sequencing (miRNA-seq), and five miRNAs were selected for validation. Potential target genes of these five miRNAs were predicted using the miRWalk3.0 database, and the overlapping portions were analyzed using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Comparison of the cases and controls revealed 127 known differentially expressed miRNAs. A total of 44 and 83 miRNAs were upregulated and downregulated, respectively. Through target gene prediction of five miRNAs, we obtained 1360 target genes. GO enrichment analysis showed that the target genes of these dysregulated miRNAs were related with secretion, protein binding, and cell growth. The KEGG pathway analysis showed that pathways in cancer, calcium signaling, and rat sarcoma (Ras) signaling, etc. were likely regulated by these five miRNAs. These findings highlight the distinct expression patterns of miRNAs in coal-burning endemic fluorosis.

Xue Han

Papers

Analysis of the microRNA Profile of Coal-Burning Endemic Fluorosis Using Deep Sequencing and Bioinformatic Approaches

miR-486-3p regulates CyclinD1 and promotes fluoride-induced osteoblast proliferation and activation.