The Role Of Let-7c-5p On Osteoblast Proliferation And Activation Induced By Fluoride Via Regulating CyclinD1

Objective:Endemic fluorosis is a chronic systemic disease caused by high fluoride intake and the main symptoms include dental and skeletal fluorosis.The activation of osteoblast is closely related to skeletal fluorosis,which tightly regulated by the cell cycle.Based on our previous findings,this study demonstrated the participation of let-7c-5p in fluoride-induced proliferation and activation of human osteoblasts by regulation of Cyclin D1 expression.The findings of this study are conducive to the control of endemic fluorosis,which open up new paths to developing effective treatments for fluorosis.Methods:1.The expression and correlation analysis of let-7c-5p,Cyclin D1 and Wnt9 a among the people exposed to fluoride: 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 case and control inclusion criteria,248 villagers were enrolled in to this study.Each participant had provided the informed consent.Morning urine and the fasting peripheral blood samples were collected to test for fluoride exposure level.Urinary fluoride(UF)was determined using a national standardized ion selective electrode approach.Based on UF levels,the participants were classified into different groups: UF<1.96 mg/g Cr(n=117);1.96 mg/g Cr≤UF<3.92 mg/g Cr(n=65);UF>3.92 mg/g Cr(n=66).The activity of alkaline phosphatase(ALP)was determined with a standard micronutrient enzyme method,and bone Gla protein(BGP)content in patient serum was measured with an ELISA Kit.Our previous study employed mi RNA-seq to measure mi RNA levels in plasma samples from coalburning endemic fluorosis patients.Here,we use bioinformatics to identify downregulated mi RNAs that target Cyclin D1,including let-7c-5p,which also targets Wnt9 a.Therefore,we selected let-7c-5p for further study.Quantitative real-time PCR(q RT-PCR)was used to verify the expression of let-7c-5p and Wnt9 a m RNA,and ELISA was used to detect the expression of Wnt9 a protein,Spearman correlation analysis was used to analyze correlations.2.Effect of Na F application on let-7c-5p,Cyclin D1,Wnt9a/β-catenin pathway expression: Human 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 Na F for 72 h.let-7c-5p,Cyclin D1 and Wnt9 a m RNA expression were detected by q RT-PCR.The expression of Cyclin D1 and Wnt9 a protein were detected by Western-blotting.β-catenin nuclear translocation was detected using Western blotting and immunofluorescence.To determine the role of β-catenin binding within the Cyclin D1 transcriptional regulatory region in Na F-induced Cyclin D1 upregulation,we conducted Chromatin immunoprecipitation(Ch IP)assays to measure β-catenin binding levels.3.let-7c-5p regulates Cyclin D1 in fluoride-mediated osteoblast proliferation and activation:(1)Human osteoblasts were transfected with let-7c-5p mimic and mimic NC for 24 h.The expression of let-7c-5p after transfection was detected by q RT-PCR to determine the success of transfection.Then,human osteoblasts were treated with 1200 μmol/L Na F for 72 h.let-7c-5p expression was detected by q RT-PCR.The effect of fluoride on proliferation and activation of osteoblasts were detected by CCK-8,Ed U and flow cytometry.ALP activity and BGP content were detected by micronutrient enzymes standard method and ELISA methods.(2)The target gene binding site(s)were predicted with bioinformatics soft.The dual luciferase reporter gene assay system to confirm that Cyclin D1 is a let-7c-5p target.Human osteoblasts were transfected with let-7c-5p mimic and mimic NC for 24 h.Then,human osteoblasts were treated with 1200 μmol/L Na F for 72 h.The expression of Cyclin D1 m RNA and protein were detected by q RT-PCR and Western blotting.(3)The target gene binding site(s)were predicted with bioinformatics soft.The dual luciferase reporter gene assay system to confirm that Wnt9 a is a let-7c-5p target.Human osteoblasts were transfected with let-7c-5p mimic and mimic NC for 24 h.Then,human osteoblasts were treated with 1200 μmol/L Na F for 72 h.q RT-PCR and Western blotting were used to measure Wnt9 a expression in human osteoblasts.Western blotting and immunofluorescence were used to measure β-catenin nuclear translocation.Ch IP assays to determine the role of β-catenin binding within the Cyclin D1 transcriptional regulatory region.Results:1.With the increase of UF concentration,the expression of let-7c-5p decreased with the increase of UF concentration.The m RNA transcription and protein expression of Wnt9 a increased.Simultaneously,according to correlation analysis,let-7c-5p showed a negative correlation with Cyclin D1 and Wnt9 a level.These findings suggest that let-7c-5p may regulate the increase in Cyclin D1 and Wnt9 a levels.2.With the increase of NaF dose,the expression of let-7c-5p decreased,while the mRNA and protein expression of Cyclin D1 and Wnt9 a increased.Western blotting and immunostaining detected the migration of β-catenin into the nucleus,which increased with Na F content.Ch IP assay demonstrated that β-catenin could bind to the transcriptional regulatory region of Cyclin D1 in osteoblasts and that Na F promoted this interaction.These results showed that Na Fmediated upregulation of Cyclin D1 functions by activating the Wnt9a/β-catenin pathway.3.(1)let-7c-5p expression in the let-7c-5p overexpression group increased compared with that in the mimic NC group,indicating that the transfection was effective.Osteoblasts transfected with let-7c-5p mimic increased the expression of let-7c-5p compared with osteoblasts exposed to fluoride.The viability of osteoblasts transfected with let-7c-5p mimic was inhibited compared with osteoblasts along under Na F treatment.Furthermore,overexpressing let-7c-5p increased the proportion of G0/G1 phase osteoblasts but decreased those in the S and G2/M phases.Osteoblast proliferation rates were also reduced,and cell cycle progression was delayed.In addition,ALP and BGP values in the let-7c-5p mimic-transfected osteoblasts were decreased.As demonstrated by these findings,let-7c-5p overexpression might suppress osteoblast proliferation and activation induced by Na F.(2)The results of dual luciferase reporter gene assay showed that let-7c-5p directly binds to Cyclin D1 3’UTR.Osteoblasts transfected with let-7c-5p mimic increased the m RNA and protein expression of Cyclin D1 decreased.These data demonstrate the involvement of let-7c-5p in the increase of Cyclin D1 expression induced by Na F in osteoblasts and indicate the post-transcriptional downregulation of Cyclin D1 by let-7c-5p in osteoblasts.(3)The results of dual luciferase reporter gene assay showed that let-7c-5p directly binds to Wnt9 a 3’UTR.Osteoblasts transfected with let-7c-5p mimic increased the expression of Wnt9 a m RNA and protein compared with osteoblasts exposed to fluoride.The accumulation of β-catenin into the nucleus decreased,and the enrichment of β-catenin in Cyclin D1 transcriptional regulatory region decreased.These findings suggest that let-7c-5p is involved in the Na F-induced upregulation of Cyclin D1 via the Wnt9a/β-catenin pathway at the transcriptional level.Conclusions:1.let-7c-5p regulates Cyclin D1 expression at post-transcriptional and transcriptional levels via the Wnt9a/β-catenin pathway,thereby participating in fluoride-induced proliferation and activation of human osteoblasts.2.let-7c-5p/Wnt9a/Cyclin D1 axis is involved in the occurrence and development of fluorosis.