The Function And Mechanism Of MiRNA-629-3p In Osteo/odontogenic Differentiation Of SCAP

BackgroundStem cells from apical papilla(SCAP)have been found as a new type of dental mesenchymal stem cell(MSCs),which were isolated from the apical papilla of immature permanent teeth.Compared with dental pulp stem cells(DPSCs),SCAP possesses a stronger proliferative and differential ability.It is reported that SCAP can differentiate into odontoblast cells and form root dentin,which plays an important role in root development.Based on these features,SCAP have been widely recognized a reliable cell candidate for tissue engineering.MicroRNA is a type of endogenous,non-coding and single-stranded RNA which plays a vital role in cell development,differentiation,migration,apoptosis and other aspects.Although miRNA accounts for only 1%of the total genome,it can regulate the expression of more than 30%of human genes.By binding to the 3’-untranslated region(3’UTR)of target messenger RNA(mRNA),miRNA could lead to the mRNA degradation or translational suppression,inhibiting the expression of target gene eventually.Root canal therapy has been the mainstream skill clinically for treating pulpal and periapical ’diseases to date.With the development of tissue engineering and regenerative medicine,the role of miRNA in pulp-dentine complex regeneration has attracted ever-growing attention.In the present study,we established the miRNAs profiling of untreated and mineralized SCAP using microarray.The differentially expressed miRNAs were detected by Real-time PCR to verify the result of microarray and then miR-629-3p was selected.The objective of this study is to identify the effect and related mechanism of miR-629-3p on osteo/odontogenic differentiation of SCAP,which can provide a theory basis to optimize the stem cell therapy mediated by miRNA.Methods1.The SCAP was isolated and cultured using enzyme digestion.Flow cytometry was performed to identify the expression of stem cell markers.After osteogenic induction for 14 and 21 days,cells were stained by ALP and Alizarin Red S.Oil Red O staining was performed after adipogenic induction for 28 days.2.After 72h treatment of normal and osteogenic-induced medium,the total RNA in cells was extracted using TRIzol reagent.The samples were sent to company for higher throughput sequencing.Through bioinformatics methods and Real-time PCR,we analyzed the differentially expressed miRNAs,and miR-629-3p was finally selected.We change the endogenous miR-629-3p expression by transient transfection of miR-629-3p mimics and inhibitor.Alizard red,Real-time PCR and Western blot were performed to compare the difference of osteo/odontogenic differentiation capacity.3.The target gene of miR-629-3p was predicted by bioinformatics software such as Targetscan and miRDB.After constructing the wild-type and mut-type vector plasmid,the luciferase assay was performed to confirm this target gene.Transferring siRNA to reduce the expression of that target gene,Alizard red staining,Real-time PCR and Western blot were performed to compare the difference of osteo/odontogenic differentiation capacity.Results1.SCAP were isolated and cultured successfully.Flow cytometry showed that SCAP were positive expression of mesenchymal stem cell surface markers(STRO-1:33.9%,CD90:99.9%,CD44:98.6%,CD24:19.2%),and negative expression of hematopoietic stem cell surface markers(CD34:0.23%,CD45:0.35%).There were more mineralized nodes when cultured in osteogenic-induced medium.The lipid droplets could be found after adipogenic induction.2.The results of microarray assay showed that the expression of 64 miRNAs had a significant difference,25 of them were up-regulated and 39 of them were down-regulated.Combined the Real-time PCR assay,miR-629-3p was selected in the following study.After overexpression of miR-629-3p,the osteo/odontogenic differentiation of SCAP was significantly inhibited,while,it was enhanced after down-regulating the expression of miR-629-3p.3.According to the results of bioinformatics software,Real time PCR and Western blot,MAPK14 was selected as the candidate target gene and confirmed by dual-luciferase reporter gene assay.After knock-downing the expression of MAPK14,osteo/odontogenic differentiation of SCAP was suppressed.Conclusion1· miR-629-3p can inhibit the osteo/odontogenic differentiation of SCAP.2.The expression of miR-629-3p was down-regulated during the osteogenic differentiation.3.Dual-luciferase reporter gene assay confirmed that MAPK14 was the direct target gene of miR-629-3p.When inhibiting the expression of MAPK14,the osteo/odontogenic differentiation of SCAP was suppressed.4.miR-629-3p could inhibit the osteo/odontogenic differentiation of SCAP by targeting MAPK14.