| Objective :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,we performed a population study of coal-burning fluorosis and in vitro experiments to explore the role of miR-4755-5p in osteoblast activation of skeletal fluorosis via targeting of Cyclin D1.We aimed to explore the role and mechanism of micro RNA(miRNA)in skeletal fluorosis and to provide evidence for the final application of miRNA in the treatment of skeletal fluorosis.Methods : 1.According to the division standard of endemic fluorosis area(GB17018-2011),Hehua Village,Zhijin County,an endemic fluorosis area in Guizhou Province was selected as the investigation area.Zhangguan Village,Anshun City,was selected as the control area.According to the subject inclusion criteria,248 villagers were enrolled into this analysis.The blood and urine samples of the subjects were collected under the principle of informed consent.Urine fluoride(UF)concentrations were determined using a national standardized ion selective electrode approach.Based on the UF content,the study population was classified into three groups: UF < 1.96 mg/g Cr(117 subjects);UF 1.96–3.92 mg/g Cr(65 subjects),and UF ? 3.92 mg/g Cr(66 subjects).The activity of alkaline phosphatase(ALP)and the content of bone Gla protein(BGP)were detected by microenzyme labeling method and enzyme-linked immunosorbent assay(ELISA)respectively.Four different algorithms from the miRWalk 3.0 database(http://mirwalk.umm.uni-heidelberg.de),including miRWalk,miRDB,miRTar Base,and Targetscan,were used to predict the miRNAs that targeted Cyclin D1.We selected candidate miRNAs according to previous miRNA-seq analyses based on three criteria: the miRNAs were downregulated and showed differential expression(P ? 0.05 and | log2FC| ? 1)in miRNA sequencing;they were predicted by at least two of the four algorithms;and the binding site was in the 3'-UTR.Quantitative real-time PCR(q RT-PCR)was used to verify the expression of miR-4755-5p,q RT-PCR was used to detect the expression of Cyclin D1 m RNA,and ELISA was used to detect the expression of Cyclin D1 protein.2.Human osteoblasts were isolated using enzyme digestion from trabecular fragments collected from healthy donors receiving surgical treatment due to fractures.Human primary osteoblasts were identified by cell morphology,alkaline phosphatase and alizarin red staining.Human primary osteoblasts were treated with 0,200,400,600,800,1000,1200,1400 or 1600 ?mol/L sodium fluoride(Na F)for 24,48,72 or 96 hours.The cell viability of primary osteoblasts exposed to fluoride was detected by CCK-8 method.Based on the results of the CCK-8 experiment,osteoblasts were treated with Na F at 0,600,or 1200 ?mol/L for 72 h for for subsequent experiments.The distribution of cell cycle was detected by flow cytometry,ALP activity and BGP content were detected by micronutrient enzymes standard method and ELISA methods.miR-4755-5p expression was detected by q RT-PCR.The expression of Cyclin D1 m RNA and protein were detected by q RT-PCR and Western blotting.3.The target binding between miR-4755-5p and Cyclin D1 was verified by double luciferase reporter gene assay.Human osteoblasts were transfected with miR-4755-5p mimic and mimic NC for 24 hours.The expression of miR-4755-5p after transfection was detected by q RT-PCR to determine the success of transfection.Furthermore,human osteoblasts were treated with 1200 ?mol/L Na F for 72 hours.miR-4755-5p expression was detected by q RT-PCR.The expression of Cyclin D1 m RNA and protein were detected by q RT-PCR and Western blotting.Cell viability was detected by CCK-8 method,cell cycle distribution was detected by flow cytometry,ALP activity and BGP content were detected by micronutrient enzymes standard method and ELISA methods.Results:1.The results showed that among the people exposed to fluoride,the ALP activity and BGP content of the high UF group increased compared with the low UF group(P all <0.05).(P all < 0.05).The results showed that the activation of osteoblasts induced by fluoride exposure was observed in the population exposed to fluoride.Based on the previous miRNA-seq results and the criteria described above,miR-4755-5p targeting Cyclin D1 were identified among downregulated miRNAs using bioinformatics software.In the fluoride-exposed population,the results showed that compared with the low UF group,the miR-4755-5p expression was reduced in the high UF group,while the m RNA transcription and protein expression of Cyclin D1 increased gradually(P all <0.05).Simultaneously,according to correlation analysis,miR-4755-5p showed a negative correlation with Cyclin D1 level,implying that miR-4755-5p may participate in Cyclin D1 upregulation.2.With the increase of fluoride dose,the proliferation index(PI),ALP activity and BGP content of primary human osteoblasts increased gradually(P all <0.05).The results showed that Na F treatment could lead to abnormal proliferation and activation of osteoblasts.Simultaneously,with the increase of Na F dose,the expression of miR-4755-5p decreased gradually(P <0.05),while the m RNA transcription and protein expression of Cyclin D1 gene increased(P all <0.05).According to these findings,for human osteoblasts,Na F led to the inhibition of miR-4755-5p expression and elevated Cyclin D1 expression,which conformed to the findings observed in the fluoride-exposed population.3.Double luciferase reporter gene assay showed that miR-4755-5p regulated the expression of Cyclin D1 by directly targeting its 3'-UTR.miR-4755-5p expression in the miR-4755-5p overexpression group increased compared with that in the negative control group(P < 0.05),indicating that the transfection was effective.Osteoblasts transfected with miR-4755-5p mimic increased the expression of miR-4755-5p compared with osteoblasts exposed to fluoride(P < 0.05).Increased Na F-induced Cyclin D1 protein was inhibited in cells treated with miR-4755-5p mimic(P all < 0.05),whereas Cyclin D1 m RNA expression was not significantly changed(P > 0.05).The results showed that overexpression of miR-4755-5p down-regulated the level of Cyclin D1 protein in osteoblasts under the action of Na F,which confirmed the regulation of Cyclin D1 expression by miR-4755-5p.After over-expressing of miR-4755-5p,the proliferation index(PI),ALP activity and BGP content of primary human osteoblasts decreased(P all< 0.05).These results suggest that overexpression of miR-4755-5p can reduce the expression of Cyclin D1 protein in human osteoblasts under the action of Na F,and then inhibit the proliferation and activation of human osteoblasts.However,in this study,the effect of overexpression of miR-4755-5p on Cyclin D1 m RNA was not observed,which means that miR-4755-5p may inhibit the expression of Cyclin D1 by inhibiting the translation of its protein rather than m RNA degradation.Conclusion:1.Fluoride exposure could induce the downregulation of miR-4755-5p.2.Downregulation of miR-4755-5p promotes fluoride-induced osteoblast activation via increasing Cyclin D1 expression.3.miR-4755-5p directly binds to the 3'-UTR of Cyclin D1 and affects its protein expression. |
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