| Objective:The abnormal proliferation and activation of osteoblasts induced by fluoride is related to skeletal fluorosis,while the mechanism has not been fully elucidated.As an important epigenetic regulator,microRNAs participate in the regulation of bone metabolism and cell cycle.Based our previous miRNA-seq results and bioinformatics analysis,in this study,we explored miR-486-3p-mediated regulation of CyclinD1 and its contribution to fluoride-induced osteoblast proliferation and activation.This study,thus,contributes significantly in sheding new light on endemic fluorosis treatment.Methods:1.Expression of miR-486-3p,CyclinD1 and TGF-β1 and correlation analysis in people exposed to fluorosis caused by coal burning: a total of 248 participants were enrolled,including 141 from Hehua Village in Zhijin County of Guizhou Province.This is a typical region subjected to coal-burning endemic fluorosis,determined as per the endemic fluorosis area classification(GB17018-2011,China).One hundred and seven villagers who did not suffer from endemic fluorosis were from Zhangguan Village in the Anshun City of Guizhou Province.Blood and urine samples were taken under the principle of informed consent.According to the urinary fluoride(UF)levels determined in the previous study,the subjects were divided into three groups:UF < 1.96 mg/g Cr group(n = 117),1.96 mg/g Cr ≤ UF < 3.92 mg/g Cr group(n =65),and UF ≥ 3.92 mg/g Cr group(n = 66).Using four different algorithms of miRWalk3.0 database(miRDB,miRWalk,Target Scan and miRTar Base)to predict miRNA,combined with the previous miRNA-seq results and screening criteria,we predicted that miR-4755-5p,let-7c-5p,and miR-486-3p were down-regulated and targeted to CyclinD1.In this paper,miR-486-3p was selected for further study.Further bioinformatics analysis showed that miR-486-3p also targeted TGF-β1 in the TGF-β1/Smad pathway.CyclinD1 expression in PBMCs as well as the ALP activity and BGP content in the serum have been detected in our previous study.qRT-PCR was used to verify the expression of miR-486-3p in plasma and TGF-β1 mRNA in PBMCs.ELISA was used to detect the protein expression of TGF-β1 in plasma.Correlations between miR-486-3p and CyclinD1,TGF-β1 were assessed using Spearman’s correlation.2.The effect of NaF on miR-486-3p,CyclinD1 and TGF-β1/Smad2/3/CyclinD1 axis:Human primary osteoblasts were isolated using enzyme digestion method and identified by morphological observation,alkaline phosphatase and alizarin red staining.Based on our former results,we treated osteoblasts similarly with either 0,600,or 1200 μmol/L NaF for 72 h.Levels of miR-486-3p,CyclinD1 and TGF-β1mRNA expression were detected by qRT-PCR.The protein expression of CyclinD1 and TGF-β1 were detected by Western blotting,which also was used to detect the expression and phosphorylation level of Smad2/3 inside and outside the nucleus.Smad2/3 nuclear translocation was detected using immunofluorescence.And the enrichment of Smad2/3 in CyclinD1 transcriptional regulatory region was detected by chromatin immunoprecipitation(ChIP)method.3.The mechanism of miR-486-3p regulating CyclinD1 in the proliferation and activation of human osteoblasts induced by fluoride:(1)miR-486-3p participates in fluoride-induced osteoblast proliferation and activation.On the basis of the confirmation of fluoride-induced proliferation and activation of human primary osteoblasts in the early stage of our research,human osteoblasts were transfected with miR-486-3p mimic for 24 h and then treated with 1200 μmol/L NaF for 72 h.qRT-PCR was used to verify the expression of miR-486-3p.CCK-8,Ed U,flow cytometry,ALP activity and BGP content were used to detect the effect of miR-486-3p on the proliferation and activation of osteoblasts.(2)miR-486-3p can directly target CyclinD1 to regulate the upregulation of fluoride-induced CyclinD1 at the post-transcriptional level.After miR-486-3p mimic was transfected into human osteoblasts for 24 h,osteoblasts were treated with 1200 μmol/L NaF for 72 h to detect the expression of CyclinD1.At the same time,the relationship between miR-486-3p and CyclinD1 was verified by the dual-luciferase reporter experiment in 293 T cells.(3)miR-486-3p can regulate CyclinD1 induced by fluoride at the transcriptional level through TGF-β1/Smad2/3 pathway.Dual-luciferase reporter gene assay was used to verify the combination between miR-486-3p and TGF-β1.After transfection of human osteoblasts with miR-486-3p mimic for 24 h,human osteoblasts were further treated with 1200 μmol/L NaF for 72 h to detect the expression of TGF-β1,while the expression,phosphorylation and translocation of Smad2/3,and the enrichment of Smad2/3 in CyclinD1 transcriptional regulatory region were detected.4.Multiple linear regression analysis of CyclinD1 expression and related miRNAs in the blood of 248 subjects: CyclinD1 mRNA and protein expression were taken as dependent variables,and miR-4755-5p、let-7c-5p and miR-486-3p expression level as independent variables to conduct multiple linear regression analysis.Results:1.Based on the previous miRNA-seq results and screening criteria,bioinformatics software was used to select miR-486-3p targeting CyclinD1 and TGF-β1 from the downregulated miRNA for follow-up experiments.Compared with the low UF group,the expression of miR-486-3p decreased and TGF-β1 expression increased in the high UF group(P all < 0.05).At the same time,the level of miR-486-3p was negatively correlated with the expression of CyclinD1(P < 0.05),and negatively correlated with the expression of TGF-β1(P < 0.05),suggesting that miR-486-3p may be involved in the upregulation of CyclinD1 and TGF-β1.2.In the experiment of human osteoblasts treated with sodium fluoride,with the increase of fluoride dose,the expression of miR-486-3p decreased gradually,while the expression of CyclinD1 and TGF-β1 increased gradually.And the expression and phosphorylated Smad2/3 protein increased and transferred more to the nucleus,and the binding of Smad2/3 to CyclinD1 transcriptional regulatory region increased(P all< 0.05).It is suggested that NaF can induce the level of miR-486-3p and increase the level of CyclinD1,and NaF can activate TGF-β1/Smad2/3/CyclinD1 axis.3.Under NaF exposure,the osteoblasts transfected with miR-486-3p mimic were compared with the osteoblasts only treated with 1200 μmol/L NaF:(1)the expression of miR-486-3p was increased,while the proliferation activity of human osteoblasts was inhibited and the progress of cell cycle was blocked(P all < 0.05).At the same time,the activity of ALP and the content of BGP were decreased(P all < 0.05).It is suggested that the overexpression of miR-486-3p can reverse the proliferation and activation of osteoblasts induced by fluoride.(2)the expression of CyclinD1 was decreased(P all < 0.05).The result of the dual-luciferase reporter assay confirmed that CyclinD1 was the target gene of miR-486-3p(P < 0.05).It is suggested that miR-486-3p can directly target CyclinD1 to regulate the upregulation of CyclinD1 induced by fluoride at the post-transcriptional level.(3)the result of the dual-luciferase reporter assay confirmed that TGF-β1 was another target gene of miR-486-3p(P < 0.05).The expression of TGF-β1,Smad2/3 and p-Smad2/3decreased while the nuclear translocation of Smad2/3 protein decreased.And the direct binding of Smad2/3 to CyclinD1 transcriptional regulatory region was inhibited(P all < 0.05).That is,the overexpression of miR-486-3p reverses the activation of TGF-β1/Smad2/3/CyclinD1 axis induced by NaF,indicating that miR-486-3p can regulate the up-regulation of CyclinD1 induced by fluoride at the transcriptional level through TGF-β1/Smad2/3 signal pathway.4.Multiple linear regression analysis showed that there was a negative linear regression relationship between the expression of CyclinD1 and the level of miR-4755-5p,let-7c-5p and miR-486-3p in 248 subjects,and suggested that miR-486-3p had the greatest effect on the expression of CyclinD1 mRNA and protein in the three miRNA.Conclusions:miR-486-3p regulates CyclinD1 expression at post-transcriptional and transcriptional levels via the TGF-β1/Smad2/3 signaling pathway,thereby participating in fluoride-induced proliferation and activation of human osteoblasts. |
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