The Role And Mechanism Of MiR-497-5p In Osteo/Odontogenic Differentiation Of Stem Cells From Apical Papilla

BackgroundCaries and dental trauma are common and frequently-occurring diseases in adolescents.If timely and effective treatment is not supplied,it will lead to pulpal necrosis and periapical lesions,causing stagnant teeth development in young permanent teeth and corresponding resistance is weakened.It will seriously affect the masticatory function and aesthetics of adolescents.Human SCAP are present in the developing apical papilla tissue of permanent teeth.Studies have shown that SCAP play an important role in root development.Because of its stronger proliferation and differentiation potential than dental pulp stem cells,it becomes an important seed cell for dental pulp dentin regeneration.MicroRNA(miRNA)is a kind of non-coding small RNA,which can bind to the 3’UTR of the target mRNA and negatively regulate the expression of its target genes.They play a vital role in post-transcriptional regulation.Recently,it has been found that miRNAs have important effects on the osteogenic differentiation of mesenchymal stem cells by regulating signaling pathway.However,it is still unclear how miRNA regulate the osto/odontogenic differentiation of SCAP.In this study,the miRNAs expression profiles were established related to SCAP osto/odontogenic differentiation.According to bioinformation analysis and qPCR,we chose miR-497-5p for the further study,which was significantly elevated in osteogenic/dentinogenic differentiation of SCAP.The role and mechanism of miR-497-5p in osteo/odontogenic differentiation of SCAP were:investigated.This study will provide experimental basis for pulp dentin regeneration.It also can lay the foundation for tissue regeneration involved in miRNA and provide new therapeutic strategy for periapical lesions of immature permanent teeth.Methods and resultsPart Ⅰ Culture and identification of human SCAPMethodsSCAPs were isolated and cultured using the enzyme digestion.CCK-8 assay was used to analyzed the absorbance value of SCAP for 10 days and the growth curve was drawn.Crystal violet staining was performed to detect the ability of cell cloning.The mesenchymal stem cell markers were detected by flow cytometry analysis.To investigate the multi-directional differentiation potential of SCAP,we tested the alkaline phosphatase staining,alizarin red staining and oil red O staining after osteogenesis and adipogenic induction.Results.Cells grew in clusters and mostly in long spindle shape after 7-10 days.CCK-8 assay showed that the growth curve of SCAP was appeared "S" type which included incubation period,logarithmic growth period and plateau period.The cell proliferation was observed,and the colony formation efficiency was about 39%based on crystal violet stanning.Flow cytometric analysis revealed expression of mesenchymal stem cell surface markers STRO-1(31.8%)、CD24(23.2%)and CD146(99.5%).Meantime,hematopoietic surface marker CD34(0.27%)and CD45(0.16%)were negatively expressed.ALP,alizarin red and oil red O staining demonstrated that SCAP could differentiate into osteoblasts/adipocytes.Part Ⅱ Screening the differentially expressed miRNAs and determining targetmiR-497-5p during osteo/odontogenic differentiation of SCAPMethodsMicroarray analysis(miRNA 4.0,Affymetrix)was performed to screen differentially expressed miRNAs between control and osteogenic induced SCAP.qPCR were used to confirm the results,and target miRNA was picked.The expression phase of miR-497-5p during osteogenic differentiation of SCAP was detected by qPCR.ResultsThe cluster analysis showed that 64 miRNAs changed after osteogenic induction(≥2.0 fold).Among them,25 miRNAs were up-regulated and 39 miRNAs were down-regulated.Five miRNAs(hsa-miR-660-5p,hsa-miR-497-5p,hsa-miR-181a-2-3p,hsa-miR-3651,hsa-miR-6511a-3p)were detected to validate the chip results by qPCR.The expression of miR-497-5p was significantly increased in SCAP osteo/odontogenic differentiation,consistent with the chip.The expression level of miR-497-5p decreased on the third day,but there was no statistical significance.After that,the expression level was significantly up-regulated from day 7,and reached the highest on the day 21 day.Part Ⅲ Effects of miRNA-497-5p on osteo/odontogenic differentiation of SCAP MethodsAfter changing the endogenous expression of miR-497-5p by using transfection of miR-497-5p mimics or inhibitor,ALP activity was detected,ALP staining,alizarin red staining,qPCR and western blot were performed to detected the osteo/odontogenic differentiation capacity.ResultsAfter enhancing the expression level of miR-497-5p,ALP activity was increased,ALP staining dyed deeper blue purple and alizarin red staining showed more mineralized nodules.qPCR and western blot analyses showed that the osteo/odontogenic differentiation of SCAP were enhanced.On the contrary,it was suppressed after decreasing the expression of endogenous miR-497-5p.Part Ⅳ The mechanism study of miRNA-497-5p in osteo/odontogenicdifferentiation of SCAPMethodsBioinformation analysis and dual-luciferase reporter gene assay were used to confirm the target gene of miR-497-5p.SiRNA was used to knocked down the expression level of Smurf2.ALP activity was detected,ALP staining,alizarin red staining,qPCR and western blot were performed to detected the osteo/odontogenic related genes expression.The expression level of TGF-β/Smad signaling pathway related genes were analyzed after transfected miR-497-5p mimics or inhibitor as well as knockdown of Smurf2.Western blot was used to detect the changes of osteo/odontogenic genes after inhibiting endogenous miRNA-497-5p and interfering Smurf2.ResultsDual-luciferase reporter gene assay showed that Smurf2 was the direct target gene of miR-497-5p.The osteo/odontogenic differentiation capacity was enhanced after knockdown of Smurf2.The expression of Smad2,Smad3 and Smad4,the important factors of TGF-β/Smad signaling pathway,increased significantly after over-expression of miR-497-5p,while the expression of Smad2,Smad3 and Smad4 decreased significantly after inhibiting endogenous miR-497-5p.After silencing Smurf2,the mRNA and protein levels of Smad2,Smad3 and Smad4 were significantly increased.We also found that the interference with Smurf2 could hinder the effects of miR-497-5p inhibitor on osteo/odontogenic differentiation.Conclusion1.We first discovered that miR-497-5p can promote osteo/odontogenic differentiation of SCAP.2.Dual-luciferase reporter gene assay showed that Smurf2 was the direct target gene of miR-497-5p.3.We demonstrate that Smurf2 can inhibit the osteo/odontogenic differentiation of SCAP.4.miR-497-5p regulates TGF-β/Smad signaling pathway via targeting Smurf2 and promotes osteo/odontogenic differentiation of SCAP.