The Screening And Functional Study Of Key Circular RNA In Odontogenic Differentiation Of Human Dental Pulp Stem Cells

BackgroundPulp disease and periapical disease are the most common oral diseases.The main treatment method is to remove the diseased pulp and repair the tissue defect with synthetic materials.However,these materials have limitations and cannot fully restore the function of tissues.With the development of tissue engineering regenerative medicine,pulp regeneration has become an ideal treatment.Human dental pulp stem cells?hDPSCs?are the most commonly used seed cells and play an important role in tooth pulp regeneration.Although many scholars have been studying how to achieve the odontogenic differentiation of hDPSCs,the exact molecular mechanism has not been obtained.Therefore,if a new biological marker can be found to reveal the molecular mechanism of the odontogenic differentiation of hDPSCs,it is of great significance for pulp regeneration.Circular RNA?circRNA?is a special non-coding RNA.It has been found that circRNA is not only widely distributed,highly conserved and specifically expressed in tissue cells,but also can regulate the pluripotency and differentiation ability of embryonic stem cells.Therefore,circRNA is considered by scholars to be an ideal disease diagnostic marker and therapeutic target,with important research significance and clinical application value.However,there are few reports on circRNA in the process of odontogenic differentiation of hDPSCs.The purpose of this study is to study the effect of circRNA on the odontogenic differentiation of hDPSCs,so as to provide a new idea for pulp regeneration in the futureAim1.The expression levels of circRNA before and after the mineralization of hDPSCs were determined2.Bioinformatics was used to analyze the gene chip results and construct the circRNa-miRNA-mRNA network map3.To preliminarily explore the influence of hsa circ 0001599 on the odontogenic differentiation of hDPSCsMethods1.HDPSCs were isolated and cultured by modified enzyme tissue block digestion,and their biological characteristics were identified.2.CircRNA expression levels in hDPSCs before and after mineralization induction were analyzed by gene chip technology,and the results were verified by quantitative real-time polymerase chain reaction?qRT-PCR?technology.Then,bioinformatics method was used to analyze the results of gene chip,and a network of circrna-mirna-mrna competing endogenous RNAs?ceRNA?was constructed to screen out the key circRNA in the process of odontogenic differentiation of hDPSCs.3.Construct hsa_circ₀001599 shRNA lentivirus to silencing its expression and detect its ability to odontogenic differentiation of hDPSCsResult1.HDPSCs were isolated and cultured by modified enzyme tissue block digestion,and the ability to clone was detected by monoclonal experiment.CCK8 reagent was used to detect its good growth condition.The induction experiments of mineralization,fatogenesis and chondrogenesis show that it has the ability of multidirectional differentiation.The flow detection results showed that the positive expression of mesenchymal stem cell surface markers CD29?99.96%?,CD44?99.99%?,CD90?99.99%?and stro-1?18.39%?2.12929 differentially expressed circRNAs were screened by microarray results,of which 43 were up-regulated and 144 were down-regulated?variation multiple?1.5 or?-1.5,P<0.05?.Qrt-pcr results were consistent with microarray results.Bioinformatics results showed that the target genes of significantly differentially expressed circRNAs are related to multiple biological processes such as cell communication and signal transduction,and participate in the regulation of signaling pathways such as Wnt and TGF-?.The results of ceRNA network diagram indicate that there may be a potential regulatory relationship between differentially expressed circRNA and miRNA and odontogenic differentiation related mRNAs3.The lentivirus sh-circ 1599 was successfully constructed and transf'ected into the cells.The ALP staining and alizarin red staining results showed that after 14 days of mineralization induction,the ALP activity of the cells in the hsa circ 0001599 expression inhibition group decreased,and the mineralization nodules decreased qRT-PCR and Western Blot results showed that silencing the expression of hsa circ 0001599 inhibited the expression of odontogenic differentiation related genes DSPP?Dentin sialophosphoprotein??DMP-1?Dentinmatrixprotein??ALP?Alkaline phosphatase??OCN?Osteocalcin?Conclusion1.In this study,cells isolated and purified from human dental pulp by tissue block enzyme digestion showed high expression of mesenchymal stem cell surface markers,which proved to be the source of mesenchymal.With the ability of clone formation and multidirectional dfferentiation,it can be identified as hDPSCs.2.It is true that a large number of circRNA are significantly differentially expressed in the process of odontogenic differentiation of hDPSCs.Preliminary bioinformatics analysis showed that differentially expressed circRNA target genes were involved in various signal pathways related to odontogenic differentiation of hDPSCs,and there might be a circrna-mirna-mrna regulatory network.3.When the expression of hsa circ 0001599 was silenced,the odontogenic ability of hDPSCs was inhibited,proving that hsa_circ₀001599 could promote the odontogenic differentiation of hDPSCs.