MiR-23a Effects On Osteogenic Activity Of Fluoride Treated UMR-106 Cells

Objective:To screen and validate differential expression of miR-23a in UMR-106 cells treated with NaF.To construct over expression vector and antisense oligonucleotide for miR-23a,further to explore the role of miR-23a on osteogenic activity by fluoride-induced.Methods:1.Screening and verification of the differential miRNA in UMR-106 cells with NaF treated.UMR-106 cells were treated with 2×10~3?mol/L NaF,RNA samples were analyzed by using miRNA array,the miR-23a differential expression was validated by Real-time PCR.To predict the target gene of miR-23a and its biological function and signaling pathway by bioinformatics analysis.2.Effects of miR-23a treated with NaF on UMR-106 cells of osteogenic activity.(1)Effects of NaF on osteogenic activity in UMR-106 cells.UMR-106 cells were treated with 0,5×10~2,10~3 m2×10~3?mol/L NaF for 24h,48h and 72h respectively.ALP,Osx BMP-2,Co L I,BGP,Runx2 mRNA expression were detected by real-time PCR.The protein expression of CoL I,Runx2,Osx were detected by Western blot.Disodium phenyl phosphate was used to detect ALP activity.(2)The role of miR-23a in the changes of osteogenic activity of fluoride exposed in UMR-106 cells.The effect of miR-23a overexpression and miR-23a oligonucleotide on osteogenic activity of UMR-106 cells.UMR-106 cells were respectively transfected by miR-23a overexpression vector and miR-23a oligonucleotide,then cultured in the NaF for 48h.Real-time PCR was performed to examine the gene expression of ALP,BMP-2,CoL I,BGP,Runx2 and Osx.Western blot was performed to examine the protein expression of COL I,Runx2 and Osx.3.Verification of the target gene of miR-23a.The expression of target gene for miR-23a were detected by Real-time PCR and Western blot.The luciferase reporter assay was used to validate the target gene of miR-23a.Results:1.Screening and verification of the differential mi RNA in UMR-106 cells treated with fluoride.The mi RNA array analysis identified 10miRNAs differentially expression.The variation trend of miR-23a expression by real-time PCR was consistent with that of expression profiles.Bioinfirmatics analysis reveals that the target genes of miR-23a were associated with osteoblast differentiation,MAPK and FoxO pathways.2.Effects of miR-23a treated with Na F on UMR-106 cells'osteogenic activity.(1)The effect of Na F on osteogenic activity in UMR-106 cells.(1)UMR-106 cells were cultured with NaF for 24h;The expression of BMP-2 mRNA was increased in low dose group than that in the control group.the expression of BGP mRNA in fluoride treated groups were higher than that in the control group.The expression of ALP,Runx2 and Osx mRNA in medium and high fluoride groups were dreased compared with control group.(2)The expression of ALP,Col I,BMP-2 and Runx2 mRNA in medium and high fluoride groups were lower than those in the control group with NaF for 48h.The expression of Osx mRNA in high fluoride group was lower than that in the control group.(3)After treating UMR-106cells with NaF for 72h,the expression of ALP,BGP,Runx2 and Osx mRNA in low fluoride groups were higher than those in the control group.The expression of ALP,Col I,BMP-2,Runx2 and Osx mRNA in medium and high fluoride groups were lower than those in the control group..(4)The expression of ALP activity of UMR-106 cells in 3 fluoride groups was higher than that in the control group treated NaF for 24h,the activity of ALP in cells and cell culture medium elevated in medium fluoride group for fluoride treated 48h and 72h.(5)Compared with the control group,the protein expression of Col I and Runx2 in high fluoride groups were decreased with NaF for 24h and 72h.The protein expression of Osx was decreased in high fluoride groups for 72h with NaF.The protein expression of Col I was decreased with the increase of NaF concentration.The expression of Runx2 protein was lower in high fluoride groups than that in each dose group for 48h with NaF.(2)The role of miR-23a in the changes of osteogenic activity with fluoride exposed UMR-106 cells.(1)The effect of miR-23a overexpression on osteogenic activity of UMR-106 cells.The expression of ALP,Col I,BMP-2 mRNA,and Col I,Runx2 protein in pcDNA3.1/mir-23a group were lower than those in pcDNA3.1 group.Runx2,BGP mRNA were higher than pcDNA3.1 group.Compared with NaF group,the Col I mRNA was lowed in pcDNA3.1/miR-23a/NaF group.(2)The effect of miR-23a oligonucleotide on osteogenic activity of UMR-106 cells.The expression of ALP mRNA and Col I protein in anti-miR-23a group were higher than that in anti-NC group.Compared with Na F group,the BGP and Runx2 mRNA were higher in anti-miR-23a/NaF group.3.Verification of the target gene of miR-23a.(1)Compared with anti-NC group,the Rap1b mRNA was decreased in anti-miR-23a group.(2)The luciferase activity of wild-type plasmid of Rap1b with miR-23 was not found significantly different between wild-type plasmid of Rap1b with mimics NC.Conclusions:1.The miR-23a was up-regulated in NaF group in UMR-106 cells.The target genes of miR-23a were found possibly to be involved with the osteoblast differentiation and bone metabolism,which suggested that miR-23a may participate in the functional regulation of osteoblast of NaF treated.2.NaF had a double action to ostengenic activity in UMR-106 cells,and the double action may be related to the sodium fluoride dose and combined for cytokines.3.The Col I mRNA was decreased in pcDNA3.1/miR-23a/NaF group and the BGP,Runx2 mRNA was increased in anti-miR-23a/NaF group.The results suggest that mi R-23a could play a certain role in the process of osteogenic activity expression with NaF treated.4.Dual-luciferase reporter assay system suggested that there might not have the direct targeting regulatory relations between miR-23a and Rap1b 3'UTR.