The Role Of MiR-122-5p On Proliferation And Activation Of Osteoblasts Induced By Fluoride Via Targeting CDK4

Objective: Endemic fluorosis is a chronic systemic disease caused by long-term excessive intake of fluoride,and it is one of the key endemic diseases in China.Excessive intake of fluoride can cause bone turnover disorders,which can lead to skeletal fluorosis.Abnormal proliferation and activation of osteoblasts play a key role in bone turnover disorder of skeletal fluorosis.In this study,on the basis of previous findings that the high expression of CDK4 was involved in the proliferation and activation of osteoblasts in skeletal fluorosis,bioinformatics software combined with the previous miRNA-seq results were used to predict the differential expressed miRNAs that target CDK4.To explore the role of miR-122-5p involved in fluoride-induced proliferation and activation of human osteoblasts by targeting CDK4,the relationship between miR-122-5p expression and CDK4 expression,fluoride exposure,osteoblast proliferation and activation was analyzed in population study and in vitro.The aim of this study is to explore the role of miRNA in the occurrence and development of skeletal fluorosis and to provide new ideas and directions for the prevention and treatment of endemic fluorosis.Methods 1.Based on the standard of endemic fluorosis area(GB17018-2011,China),Hehua Village,Zhijin County,an endemic fluorosis area in Guizhou Province was selected as the investigation point,and Zhangguan Village,Anshun City,as the control area.According to the case and control inclusion criteria,248 villagers were enrolled into this analysis.The blood,hair and urine samples of the participants were collected under the principle of informed consent.Urinary fluoride(UF)and hair fluoride(HF)were determined using a national standardized ion selective electrode approach.Based on UF levels,the participants were classified into three groups,as follows: UF < 1.96 mg/g Cr(n=117);UF 1.96–3.92 mg/g Cr(n=65),and UF ? 3.92 mg/g Cr(n=66).Based on HF levels,the participants were classified into four groups,as follows: HF< 0.78 ?g/g,(n=60),0.78 ?g/g? HF<1.49 ?g/g,(n=63),1.49 ?g/g? HF<3.86 ?g/g,(n=63),HF ?3.86 ?g/g,(n=62).Four different algorithms(miRwalk,miRDB,miRTar Base,and Target Scan)were used to predict miRNAs targeting CDK4 using the miRWalk 3.0database.Based on the previous miRNA-seq results of the research group,candidate miRNAs were selected based on three criteria: downregulated and differential expression(P?0.05 and | log2FC|?1)miRNAs are identified using miRNA-seq in our previous study;the selected miRNA is at least the intersection of two software;the binding site is 3'UTR.The activity of alkaline phosphatase(ALP)and the content of bone Gla protein(BGP)were detected by micronutrients enzymes standard method and enzyme-linked immunosorbent assay(ELISA),respectively.Quantitative real-time PCR(q RT-PCR)was used to detect the expression of miR-122-5p,q RT-PCR and ELISA were used to detect the expression of CDK4 m RNA and protein.2.Human primary osteoblasts were isolated by enzyme digestion method and identified by morphological observation,alkaline phosphatase and alizarin red staining.Human osteoblasts were treated with 0,200,400,600,800,1000,1200,1400 or 1600 ?mol/L Na F for 24,48,72 and 96 h.CCK-8 assay was used to detect cell vitality.According to the results of CCK-8,human osteoblasts were treated with 0,600,1200 ?mol/L Na F for 72 hours for subsequent experiment;flow cytometry was used to detect cell cycle distribution;micronutrients enzymes standard method was used to detect ALP activity;ELISA was used to detect BGP content;q RT-PCR was used to detect the expression of miRNA;q RT-PCR and western-blotting were used to detect the expression of CDK4 m RNA and protein.3.Twenty-four hours after miR-122-5p mimic and mimic NC transfection into human osteoblasts,human osteoblasts were treated with 1200 ?mol/L Na F for 72 h,and q RT-PCR was used to detect miRNA expression;q RT-PCR and western blotting were used to detect the expression of CDK4 m RNA and protein;CCK-8 assay was used to detect the cell activity of human osteoblasts;flow cytometry was used to detect the cell cycle distribution;micronutrients enzymes standard method and Elisa were used to detect ALP activity and BGP content;dual luciferase reporter gene assay was used to verify the targeted binding of miR-122-5p and CDK4.Results 1.Based on the previousmiRNA-seq results and the above criteria,the bioinformatics software was used to obtain the miR-122-5p targeting CDK4 in the downregulated miRNAs for subsequent experiments.The results showed that among the people exposed to fluoride,the ALP activity and BGP content of the high UF group and the high HF group increased compared with the low UF group and the low HF group(P all <0.05).Compared with the low UF group and the low HF group,the miR-122-5p expression was reduced in the high UF group and the high HF group(P all <0.05).and the CDK4 m RNA transcription and protein expression were increased(P all <0.05).At the same time,the expression of miR-122-5p was negatively correlated with that of CDK4,suggesting that miR-122-5p may be involved in the upregulation of CDK4.2.With the increase of the dose of fluoride,the proportion of primary human osteoblasts in S and G2/M phase increased,while the proportion of cells in G0 / G1 phase decreased,and PI,ALP activity and BGP content increased(P all <0.05).The results showed that Na F promoted the cell cycle from G1 phase to S phase,resulting in abnormal proliferation and activation of osteoblasts.With the increase of the dose of fluoride,the expression of miR-122-5p decreased gradually,while the m RNA transcription and protein expression of CDK4 increased(P all <0.05),which indicated that Na F could downregulate the expression of miR-122-5p and upregulate CDK4 expression in human osteoblasts.The results were consistent with those observed in population exposed to fluoride.3.The results of dual luciferase reporter gene assay showed that miR-122-5p directly binds to CDK4 3'-UTR to regulate the expression of CDK4.The expression of miR-122-5p in osteoblasts transfected with miR-122-5p mimic increased compared with the NC group(P <0.05),indicating that the transfection was effective.Compared with the osteoblasts treated with 1200 ?mol / L Na F,osteoblasts transfected with miR-122-5p mimic showed increased expression of miR-122-5p(P<0.05)and decreased expression of CDK4 protein(P < 0.05),and there was no significant change on CDK4 m RNA expression(P> 0.05);the proportion of osteoblasts in G0 / G1 phase increased,while the proportion in S phase and G2 / M phase decreased;PI,ALP activity and BGP content decreased(P all <0.05).The results showed that the overexpression of miR-122-5p downregulated the expressionof CDK4 protein in osteoblasts under the effect of Na F,but had no effect on m RNA level,suggesting that miR-122-5p decreased the expression of CDK4 in osteoblasts by inhibiting the translation of CDK4 protein,and then further inhibited the effect of Na F on the proliferation and activation of human osteoblasts.Conclusion: 1.Fluoride exposure could down-regulate miR-122-5p expression.2.miR-122-5p participates in the proliferation and activation of osteoblasts induced by fluoride by regulating CDK4.3.miR-122-5p directly binds to the 3'-UTR of CDK4 and affects its protein expression.