| Background:Pulp and periapical diseases that occur in young permanent teeth can lead to insufficient dentin formation,short tooth roots,and in severe cases,premature loss of teeth,which are not conducive to the development of children’s masticatory system.The fundamental way to treat such teeth is to promote dentin regeneration.Stem cells from apical papilla(SCAP)can be isolated from the apical tissue of teeth with undeveloped roots and play an important role in dentin regeneration due to their good stem cell properties.Long noncoding RNAs(lncRNAs)are non-coding RNA molecules that widely involved in various physiological processes.Existing studies have found that lncRNAs play an important role in the osteo/odontogenesis of oral stem cells,but the mechanism has not been elucidated.Therefore,in this research,we established the lncRNAs expression profiles before and after the induction of SCAP osteo/odontogenesis,and screened the lncRNAs that were differentially expressed after the induction of SCAP by R language.The objectives of this work were to examine the potential biological role of NHS-AS1 in the osteo/odontogenesis of SCAP and to illuminate its mechanisms,so as to provide a molecular theoretical basis and new therapeutic targets for dentin regeneration.Purpose:1.To investigate the effect of lncRNA NHS-AS1 on osteo/odontogenic differentiation of SCAP.2.To elucidate the molecular mechanism of lncRNA NHS-AS1 regulating the osteo/odontogenic differentiation of SCAP.Methods:1.SCAP were isolated and cultured by enzymatic tissue block method,and cell surface markers were detected by flow cytometry;alkaline phosphatase,alizarin red S staining and oil red O staining demonstrated that SCAP have multidirectional differentiation ability.2.NHS-AS1 was over-expressed in SCAP by cell transfection technology.The expression levels of osteo/odontogenesis-related genes were detected by qRT-PCR and Western Blot.The biological function of NHS-AS1 was explored.3.Bioinformatics analysis,combined with dual luciferase reporter gene assay,was used to predict and validate the binding sites of lncRNA NHS-AS1 and miR-5011-5p as well as miR-5011-5p and SMAD7.4.The regulatory effects of miR-5011-5p and SMAD7 on osteo/odontogenesis of SCAP were investigated by cell transfection techniques and functional assays.5.The relationship amongst NHS-AS1,miR-5011-5p and SMAD7 in the osteo/odontogenesis of SCAP was verified by cell co-transfection techniques,rescue assays and functional assays.Results:1.SCAP isolated and cultured in vitro highly expressed the surface markers of mesenchymal stem cells and had the ability to differentiate into osteogenic and adipogenic cells.2.LncRNA NHS-AS1 inhibited the osteo/odontogenesis of SCAP.3.NHS-AS1 could act as miR-5011-5p sponge.The osteo/odontogenesis of SCAP was enhanced after overexpression of miR-5011-5p,and miR-5011-5p could reverse the inhibitory effect of NHS-AS1.4.SMAD7 was a target gene of miR-5011-5p,and NHS-AS1 upregulated SMAD7 expression by adsorbing miR-5011-5p.5.The osteo/odontogenesis of SCAP was weakened after over-expression of SMAD7,and SMAD7 partially reversed the positive effect of miR-5011-5p on osteo/odontogenesis of SCAP.Conclusion:1.LncRNA NHS-AS1 is found to inhibit SCAP osteo/odontogenic differentiation for the first time.2.NHS-AS1 sponges miR-5011-5p to up-regulate the expression of SMAD7.3.NHS-AS1/miR-5011-5p/SMAD7 axis regulates osteo/odontogenesis of SCAP. |
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