The Study Of MiR-376a-3p Mediating Osteo/Odontogenic Differentiation Of Stem Cells From Apical Papilla

BackgroundThe tooth development is a complex biological process involved in interaction of epithelial and mesenchyme.The root development begins when the crown is about to complete.The root develops to a certain degree where it begins to erupt.It usually takes 3-5 years from the tooth eruption to the completion of root development.During this period caries and dental trauma cause dental pulp necrosis and periapical periodontitis if they cannot be treated timely and effectively,resulting in the stagnation of tooth development.The tooth in the stage show wide pulp cavity,thin root canal wall and open apex.The situation facilitate tooth fracture,bringing about serious influence on chewing function and aesthetics.Therefore,it has become an urgent problem in endodontic field to explore the biological treatment to promote the continuous development of the root and pulp dentin regeneration.Stem cells from apical papilla(SCAP)are derived from a developing tissue,named as the apical papilla,which play an important role in root development.Compared with dental pulp stem cells,SCAP have stronger proliferation capacity and differentiation potential and become an important seed cell for pulp and dentin regeneration.However,the mechanism of its proliferation and differentiation is still unclear.MicroRNA(miRNA)are a class of small endogenous non-coding single-stranded RNAs.They bind complementary sequences of target mRNA in 3’-untranslated region(3’-UTR)to downregulate the expression of target gene and negatively regulate target gene at post-transcriptional level.miRNA play an important role in osteoblast differentiation and bone tissue formation.They promote or inhibit osteoblast differentiation by targeting transcription factors and signaling pathways associated with osteoblast differentiation.Studies have shown that many miRNAs are involved in regulating the osteogenic differentiation of human odontogenic stem cells:miR-24-3p,miR-214,and miRNA-22 act as important regulatory factors in the osteogenic differentiation of human periodontal ligament stem cells(PDLSCs).MiR-32,miR-508-5p and miR-885-5p participate in regulating the osteogenic/odontogenic differentiation of dental pulp stem cells(DPSCs)through respective mechanism.However,there is little literature as regards the role of miRNA in the osteogenic/odontogenic differentiation of developing stem cells SCAP,and the mechanism is still unclear.Our previous studies found that the expression of mir-376a-3p was increased in the osteogenic/odontogenic differentiation of SCAP.This study further investigated the effect of mir-376a-3p on the osteogenic/odontogenic differentiation of SCAP and the related role,and explored the molecular mechanism of mir-376a-3p regulating the differentiation,which would provide an experimental basis for the study of pulp and dentin regeneration.Moreover,pulp dentin regeneration based on targeted miRNA or its target genes is expected to provide new ideas and therapeutic strategies for the treatment of pulp and periapical lesions in immature permanent teeth.Methods and resultsPart Ⅰ Culture and identification of human SCAPMethodsSCAP were isolated and cultured using type I collagen enzyme and dispase.The proliferation capacity of SCAP was analyzed by CCK-8 and the growth curve was drawn.The ability of cell clone formation was detected by crystal violet staining.The molecular markers of mesenchymal stem cell in.SCAP were detected by flow cytometry analysis.The osteogenic differentiation ability of SCAP was detected by alkaline phosphatase staining and alizarin red staining after the cells were cultured using the osteogenic/odontogenic medium including Des.The adipogenesis ability of SCAP was tested by oil red O staining.ResultsThe apical papilla attached to the end of the root is loosely connected with the pulp tissue,which is easy to peel off.Under the microscope,the cells in the primary culture were mostly spindle-shaped and grew in bundles or clusters.The cell proliferation was observed and the growth curve of SCAP was drawn based on the CCK-8 results.It was appeared "S" type,which included three growth periods:incubation period,logarithmic growth period and plateau period.Flow cytometric analysis showed positive expression of mesenchymal stem cell markers STRO-1(36.5%)and SCAP specific marker CD24(22.1%).Meantime,hematopoietic stem cells marker CD34(0.6%)and CD45(0.047%)were negatively expressed.ALP and alizarin red staining displayed blue-purple and a large number of brown mineralized nodules,revealing osteogenic differentiation.Oil red O staining demonstrates the formation of red lipid droplets,that is to say the cells differentiated into adipocytes.It indicates that SCAP have multidirectional differentiation potential.Part Ⅱ The effect of miR-376a-3p on osteogenic/odontogenic differentiation of SCAPMethodsThe expression of miR-376a-3p was detected by qPCR at day 0,3,7,14 and 21 after SCAP osteogenic/odontogenic medium culture.Further,miR-376a-3p mimics/inhibitor were transfected into SCAP to change the endogenous expression of miR-376a-3p,then a series of assay were carried out to investigate the influence of miR-376a-3p on osteogenic/odontogenic differentiation of SCAP.ALP activity was tested.The mRNA and protein expression of osteogenic/odontogenic genes were detected by qPCR and Western blotting.And ALP and alizarin red staining were also performed.ResultsThe expression of miR-376a-3p was increased gradually from day 3 with the time going on.The overexpression of miR-376a-3p improved ALP activity,the mRNA and protein expression of osteogenic/odontogenic genes.The ALP staining detected darker bluish violet.More mineralized nodules were revealed by Alizarin red staining.This finding indicated that enhancing the expression of miR-376a-3p promoted osteo/odontogenic differentiation of SCAP.On the contrary,the interference of miR-miR-376a-3p down-regulated ALP activity,the mRNA and protein expression of osteogenic/odontogenic genes.In addition,ALP and Alizarin red staining showed lighter bluish violet and few mineralized nodules.It suggested that the osteogenic/odontogenic differentiation of SCAP was suppressed by inhibiting the expression of endogenous miR-376a-3p.Part Ⅲ The mechanism of miR-376a-3p in osteo/odontogenic differentiation of SCAPMethodsBioinformatics analysis screened out four candidate target genes of miR-376a-3p.The candidate target genes were detected using qPCR and Western blotting after miR-376a-3p mimics/inhibitor were transfected into SCAP.AKT3 was changed mostly and was chosen as the target gene.The expression of AKT3 was measured in the osteo/odontogenic differentiation of SCAP.Then,the dual luciferase reporter assay was performed to determine whether miR-376a-3p directly targets ATK3.An AKT3 3’-UTR reporter vector of wild-type(WT)was constructed and co-transfected with miR-376a-3p mimics into HEK293T cells using Lipofectamine 2000.The mutant-type(Mut)of AKT3 was used as control.The activities of firefly and renilla luciferase were evaluated using the Dual-Luciferase Reporter Assay System.SiRNA was used to knocked down the expression level of AKT3.ALP activity was detected,ALP staining and alizarin red staining were performed,and the osteo/odontogenic related gene expression was determined by qPCR and Western blotting.MiR-376a-3p inhibitor and siAKT3 were co-transfected into SCAP,osteo/odontogenic genes were evaluated to make sure the role of miR-376a-3p in osteo/odontogenic differentiation via targeting AKT3.ResultsBioinformatics analysis and dual-luciferase reporter gene assay recognized that AKT3 was the direct target gene of miR-376a-3p.ALP activity,the expression of osteogenic/odontogenic genes were increased after AKT3 knockdown.The ALP and Alizarin red staining detected darker bluish violet and more mineralized nodules.It suggested that the osteo/odontogenic diffrentiation capacity was promoted after knockdown of AKT3.Moreover,we found that the inhibition effect of miR-376a-3p inhibitor on osteo/odontogenic differentiation was reversed by AKT3 knockdown.Conclusion1.miR-376a-3p was discovered for the first time to promote osteo/odontogenic differentiation of SCAP.2.Dual-luciferase reporter gene assay proved that AKT3 is the direct target gene of miR-376a-3p.3.AKT3 was testified to inhibit the osteo/odontogenic differentiation of SCAP.4.miR-376a-3p targeted Smurf2 and promoted osteo/odontogenic differentiation of SCAP.