Mechanism Of LncRNA H19 Regulating Human Dental Pulp Stem Cells Odontoblastic Differentiation

BackgroundSince the discovery of mesenchymal stem cells(MSCs),their self-renewal ability and multi-directional differentiation potential have attracted extensive attention in the field of regenerative medicine.Because human dental mesenchymal stem cells(hDPSCs)are derived from dental pulp tissue and are safe and easy to obtain,they are considered to be the key to the regeneration of dental pulp and dentin.In the past decade,hDPSCs have also been used in stem cell therapy,which has become the seed cell of tissue engineering and widely used in regenerative medicine.Long non-coding RNA(lncRNA)is a non-coding RNA molecule with a length of more than 200 nucleotides.A large number of studies have confirmed that it is mainly used as a miRNA sponge to regulate the expression of downstream target genes,so as to participate in regulating various physiological and pathological processes of the body.As one of the first identified lncrnas,the biological function of lncRNA H19 has attracted much attention.It has been shown that it plays an important role in tumor development and cell differentiation.At present,there are few reports about the mechanism of lncRNAs in the main human oral mesenchymal stem cells.The previous studies of our research group have confirmed that H19 can positively regulate the odontogenic differentiation process of hDPSCs in vitro,but its regulatory mechanism remains to be further elucidated.The mechanism of H19 regulating the differentiation of hDPSCs into odotoblasts can provide theoretical basis at molecular level and the new therapeutic targets.Objective(1)In vivo experiments further confirmed the function of H19 in promoting the odontogenic differentiation of hDPSCs.(2)To elucidate the molecular mechanism of H19 regulating odontogenic differentiation of dental pulp stem cells.Methods(1)The function of H19 in vivo was verified by Micro-CT scanning,Hematoxylin-Eosin(H&E)staining,Masson staining and immunohistochemistry.(2)Bioinformatics analysis combined with luciferase reporter gene experiment was used to predict and confirm the miRNA combined with lncRNA H19 and its downstream target genes.(3)The overexpression of miR-140-5p in hDPSCs was induced by cell transfection.After the induction culture,the expression level of mineralization related genes ALP,Runx2,DSPP,DMP-1 was detected by qRT-PCR and Western blot,and the biological function of mi R-140-5p was studied by Alizarin red staining.(4)The human recombinant protein BMP-2 or FGF9 were added into the induction culture medium of hDPSCs.The biological functions of BMP-2 and FGF9 were explored by qRT-PCR,Western blot and Alizarin red staining.(5)H19 overexpression vector and miR-140-5p mimics were co-transfected into hDPSCs.Rescure experiments were performed after odontoblastic differentiation to verify the antagonism effect of H19 against miR-140-5p in hDPSCs.Results(1)In vivo experiments showed that overexpression of H19 could increase the heterotopic formation of new dentin like structure of hDPSCs,and H19 could promote the odontogenic differentiation of HDPSCs in vivo.(2)Bioinformatics analysis combined with luciferase reporter gene showed that H19 serves as a miRNA sponge for miR-140-5p in hDPSCs.Upregulation of miR-140-5p can inhibit the differentiation of hDPSCs.(3)It is predicted that miR-140-5p has protential binding sites with 3’UTR of BMP-2 and FGF9 respectively through the databases,and the Dual luciferase reporter gene assay verified that they are the target genes of mi R-140-5p.MiR-140-5p could negatively regulate the expression of BMP-2 and FGF9,and the expression level of BMP-2 and FGF9 in hDPSCs increased after odontoblastic differentiation culture.Human recombinant protein BMP-2 and FGF9 were added into the induction culture medium respectively,which could promote the odontogenic differentiation of hDPSCs.(4)The H19 overexpression vector and miR-140-5p mimics co-transfection rescue experiment showed that H19 promoted the odontogenic differentiation of hDPSCs by sponging miR-140-5p.ConclusionH19 can promote the odontogenic differentiation of hDPSCs in vivo.The regulatory mechanism is: H19 regulates the expression of downstream target genes BMP-2 and FGF9 by sponging miR-140-5p,thus promoting the odontogenic differentiation of hDPSCs.