RESUMEN
Endemic fluorosis is caused by the intake of high environmental fluoride, which causes dental and skeletal fluorosis. Osteoblast proliferation and activation is closely related to skeletal fluorosis and is tightly regulated by the cell cycle. Several biological processes, including bone metabolism and osteoblast proliferation and activation, are regulated by a type of noncoding RNA called microRNAs (miRNAs). However, the understanding of miRNA functions in skeletal fluorosis is limited. Based on our previous miRNA sequencing results and bioinformatics analysis, we investigated the function of the miRNA let-7c-5p to regulate CyclinD1 in fluoride-induced osteoblast proliferation and activation. We designed population experiments as well as in vitro studies using 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, immunofluorescence, dual-luciferase reporters, and chromatin immunoprecipitation. The population-based analysis showed a decrease in let-7c-5p expression as fluoride exposure increased. In addition, let-7c-5p levels were negatively correlated with CyclinD1 and Wnt9a (another let-7c-5p target). We verified in vitro that let-7c-5p participates in the fluoride-induced proliferation and activation of human osteoblasts by directly targeting CyclinD1. Furthermore, we demonstrated that let-7c-5p regulates CyclinD1 expression via the Wnt/ß-catenin signaling pathway. This study demonstrated the participation of let-7c-5p in fluoride-induced proliferation and activation of human osteoblasts by regulation of CyclinD1 expression at the post-transcriptional and transcriptional levels.
Asunto(s)
Fenómenos Biológicos , MicroARNs , Línea Celular Tumoral , Proliferación Celular , Fluoruros/toxicidad , Humanos , MicroARNs/genética , OsteoblastosRESUMEN
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.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina , Fluoruros , MicroARNs , Proliferación Celular , Quinasa 4 Dependiente de la Ciclina/genética , Fluoruros/toxicidad , Humanos , MicroARNs/genética , OsteoblastosRESUMEN
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.
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Ciclina D1/metabolismo , Fluoruros/farmacología , MicroARNs/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Adulto , Western Blotting , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Endemic fluorosis is a serious problem in public health, affecting thousands of people. Abnormal proliferation and activation of osteoblasts in skeletal fluorosis lesions play a leading role and osteoblast proliferation is finely regulated by the cell cycle. There are a few reports on fluoride-induced DNA methylation. However, the role of DNA methylation of the cyclin/cyclin-dependent kinase (CDK)/cyclin-dependent kinase inhibitor (CKI) regulatory network in skeletal fluorosis has not been investigated. We used a population study and in vitro experiment to explore the relationship between the pathogenesis of skeletal fluorosis and methylation of Cyclin d1/CDK4/p21. The results showed a positive relationship between fluoride exposure and expression of Cyclin d1/CDK4, and a negative relationship between fluoride exposure and expression of P21. Hypermethylation of p21 was found in the fluoride-exposed population, and low expression of p21 attributed to promoter hypermethylation was confirmed in vitro. However, no changes in methylation levels of Cyclin d1 and CDK4 genes were observed in the population exposed to fluoride and NaF-treated osteoblasts. These results show that methylation of p21 gene has a significant impact on the proliferation of osteoblasts during the development of skeletal fluorosis. The present study was a first attempt to link the methylation of the Cyclin d1/CDK4/p21 regulatory network with osteoblast proliferation in skeletal fluorosis.
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Quinasa 4 Dependiente de la Ciclina/genética , Metilación de ADN/genética , Intoxicación por Flúor/genética , Fluoruros/efectos adversos , Adulto , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Preescolar , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Metilación de ADN/efectos de los fármacos , Femenino , Humanos , Masculino , Osteoblastos/efectos de los fármacos , Adulto JovenRESUMEN
Chronic exposure to fluoride continues to be a public health problem worldwide, affecting thousands of people. Fluoride can cause abnormal proliferation and activation of osteoblast and osteoclast, leading to skeletal fluorosis that can cause pain and harm to joints and bones and even lead to permanent disability. Nevertheless, there is no recognized mechanism to explain the bone lesions of fluorosis. In this work, we performed a population study and in vitro experiments to investigate the pathogenic mechanism of skeletal fluorosis in relation to methylation of the promoter of p16. The protein coded by the p16 gene inhibits cdk (cyclin-dependent kinase) 4/cdk6-mediated phosphorylation4 of retinoblastoma gene product and induces cell cycle arrest. The results showed that hypermethylation of p16 and reduced gene expression was evident in peripheral blood mononuclear cells of patients with fluorosis and correlated with the level of fluoride exposure. Studies with cell cultures of osteoblasts revealed in response to sodium fluoride (NaF) treatment, there was an induction of p16 hypermethylation and decreased expression, leading to increased cell proliferation, a longer S-phase of the cell cycle, and development of skeletal fluorosis. Further, the methylation inhibitor, 5-aza-2-deoxycytidine, reversed the p16 hypermethylation and expression in response to NaF. These results reveal a regulatory role of p16 gene methylation on osteoblasts activation during the development of skeletal fluorosis.
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Proliferación Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Osteoblastos/efectos de los fármacos , Fluoruro de Sodio/farmacología , Adulto , Enfermedades Óseas/sangre , Enfermedades Óseas/inducido químicamente , Enfermedades Óseas/genética , Enfermedades Óseas/orina , División Celular/efectos de los fármacos , División Celular/genética , Proliferación Celular/genética , Células Cultivadas , Preescolar , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Femenino , Fluoruros , Expresión Génica/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Osteoblastos/fisiología , Regiones Promotoras Genéticas/efectos de los fármacos , Fluoruro de Sodio/orina , Adulto JovenRESUMEN
Objective To observe the influence of coal-burning type of fluorosis on hypothalamic-pituitaryovary axis function and to explore possible mechanism in female rats.Methods Sixty SD rats were divided into two groups according to body weight with the method of random number table:control group and fluorosis group,30 rats in each group.Fluorosis group was feed with corn powder baked by high fluorine coal from Zhijin area.Changes of female rats' teeth during fluorine exposure were observed.After feeding for 180 days,24 h urine was collected in estrus and fluorine level was tested using fluoride ion-selective electrode; rats were executed and bone fluorine level was tested with high-temperature ashing-fluorine ion-selective electrode.Femoral artery blood was collected and serum was separated to test the contents of follicle stimulating hormone (FSH),luteinizing hormone (LH),testosterone (T),estradiol (E2) and progesterone (P) with electrochemiluminescence radioimmunoassay and gonadotropin-releasing hormone (GnRH),inhibin (INH) with enzyme-linked immunosorbent assay (ELISA),respectively.Organs,including hypothalamus,pituitary gland and ovary were weighted,and organ coefficients were calculated.Pathological morphology of hypothalamus,pituitary gland and ovary was observed after staining and ultrastructure of ovary was examined by electron microscopy.Results Coal-burning induced fluorine poisoning rat model was established successfully.There were no significant differences statistically in organ coefficients between fluorosis groups (0.032 ± 0.004,0.014 ± 0.008,0.037 ± 0.009) and controls (0.035 ± 0.005,0.012 ± 0.006,0.035 ± 0.004,t =0.46,0.87,0.64,all P ＞ 0.05).Rats serum GnRH,FSH,LH and T levels [(21.654 ± 4.765),(29.580 ± 5.221),(53.988 ± 6.506),(23.962 ± 2.255)μg/L] of fluorosis groups were all higher than those of controls [(10.384 ±2.250),(19.217 ± 4.743),(30.314 ± 4.443),(7.883 ± 1.973)μg/L,t =6.762,4.646,9.503,16.971,all P ＜ 0.05].But the level of P,INH [(12.635 ± 3.841),(18.926 ± 3.465)μg/L] were all lower than those of controls [(21.045 ±4.768),(48.076 ± 3.525)μg/L,t =4.344,18.649,all P ＜ 0.05].Serum E2 levels of control group and fluorosis group were (35.375 ± 10.662) and (27.500 ± 12.783)μg/L,respectively.The difference between groups was not statistically significant (t =1.821,P ＞ 0.05).No pathological changes were observed in the two groups of female hypothalamus,pituitary tissue by light microscopy and electron microscopy.Under light microscope,in the control group of normal ovarian tissue,more corpus luteum and different developmental stages of follicles were seen,granulosa cells were neatly arranged in a monolayer or multilayer.In fluorosis group,severe edema of ovarian interstitial cells and follicle degeneration increased.Cell structure and cell contours were blurred and unclear with occasional mature follicles.Under transmission electron microscope,in control group,normal ovarian granulosa cell ultrastructure was observed,nuclei were round,nuclear chromatin was uniform distributed,cytoplasm was rich in mitochondria and endoplasmic reticulum,and normal morphology.In fluorosis group,granulosa cells and interstitial cells showed apoptotic characters,such as nucleoli disappearing,mitochondrial swelling and chromatin aggregating at the nuclear membrane.Conclusions Fluorosis can induce ovarian tissue apoptosis,severely damage the micro environment.Reduction of P and INH affects ovarian,maturation and ovulation and leads to secretion of GnRH,FSH and LH.Fluorosis caused by coal-burning may induce the injury of ovary and cause abnormal secretion of hypothalamic-pituitary-ovary axis.Fluorosis has affected parts of female axis which may not be in the hypothalamus,pituitary,but causes ovarian tissue damage.
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Objective To explore the correlation between myeloperoxidase (MPO) genetic variation and coal-burning endemic fluorosis, and to understand the influence of integrated intervention including stove changes and health education on people’s health in the area. Methods In 2007, coal-burning endemic fluorosis disease areas were selected in Bijie City, Guizhou Province. No stove changes in Yachi Town, 150 patients with dental fluorosis were selected as fluorosis non-intervention group, and the intervention group was 150 patients in Changchun Town where the stoves were changed 2 years ago. The population in control group was selected in an area with non-endemic fluorosis in Changshun County. The mRNA expressions of MPO in leukoxytes were detected by real-time PCR. HepG2 cells were cultured in vitro and divided into four groups: pGL3-A group, pGL3-G group, pGL3-Control group and pGL3-Basic group. pGL3-A and pGL3-G were recombinant plasmid, while pGL3-Basic as a blank control and pGL3-Control as a positive one. The internal reference plasmid pRL-TK co-transfected the HepG2 cells with pGL3-G, pGL3-A, pGL3-Basic and pGL3-Control, respectively. The influence of sudden change of MPO gene promoter on the gene transfection activity was evaluated by a dual luciferasereporter gene system. Results The expression level of MPO mRNA in peripheral blood leukocytes in non-intervention group(0.054 ± 0 . 003 ) were higher than control and intervention groups (0.019 ± 0.004,0.019 ± 0.003, all P0.05). After the MPO-463G/A locus genetic variation occured, the luciferase reporter gene expression level of the recombinant plasmid pGL3-G(0.753 4 ± 0.086 6) was higher than that of the pGL3-A(0.490 0 ± 0.022 3, P < 0.05). Conclusions The study on MPO gene promoter-463G/A locus has prompted that MPO gene allele may be a protective factor to coal-burning fluorosis. The integrated interventions have a role in the prevention and treatment of endemic fluorosis.
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Objective To study the change of bone ?-carboxyglutamic acid containing protein(BGP) in serum of rats at the initial stage of skeletal fluorosis caused by fluoride in coal.Methods SD rats were randomly divided into 6 groups (the number of female and male in each group was the same respectively):the control group,the low-dose fluoride group,the middle-dose fluoride added nutrition group,the middle-dose fluoride group,the high-dose fluoride added nutrition group,the high-dose fluoride group.All rats in the experimental groups were fed on the corn collected from the prevalent areas and contained different contents of fluoride respectively for 90-100 days.Content of fluoride in the urine,bone,kidney,BGP in serum,bone mineral density (BMD)and calcium in the bone and urine were determined.Results The fluorosis of the rats became more serious as fluoride intake increased.On the condition of same fluoride intake,the fluorosis could be relieved if nutrients added.BGP in serum of rats in each experimental groups had a increase trend,at the earlier stage,BGP of the high-dose group was higher than that of the control group (P