| Research background:Trauma,dental caries,and improper tooth preparation can all result in pulp exposure,which can cause the infection and pain of pulp.Currently,the most common treatment for dental pulp injury is root canal treatment.The maintenance or regeneration of functioning dental pulp is essential for tooth preservation because teeth that have had root canal treatment are more susceptible to fracture as a result of the loss of dental pulp tissue.Stem cell-based dental pulp regeneration has become a promising topic of clinical research.Stem cells from apical papilla(SCAP)are important sources of cells for root development and pulp dentin regeneration.They are suitable for use as seed cells in tooth tissue engineering because of their high proliferative and differentiation potential.Long non-coding RNAs(lncRNAs)are non-coding RNAs with transcript lengths greater than 200nt.Numerous studies have demonstrated that lncRNAs are crucial in regulating the proliferation and differentiation of dental tissue-derived mesenchymal stem cells.However,the role of lncRNAs in osteo/odontogenic differentiation of SCAP has not been clarified,and more research is needed to understand their mechanism and functional model.In the prior study,our team established the expression profile of lncRNAs before and after SCAP mineralization induction and screened out the differentially expressed lncRNAs.In this study,lncRNA LINC00704 was chosen as the research object after qRT-RCR was utilized to validate the screening results.We subsequently explored its role in osteo/odontogenic differentiation of SCAP and explained the underlying molecular mechanism,providing a new therapeutic target for dental pulp dentin regeneration.Research methods:(1)SCAP were isolated and cultured by tissue enzymatic digestion,and the biological characteristics,including cell clone formation rate,cell surface markers,osteogenic differentiation and adipogenic differentiation ability,were separately confirmed by crystal violet staining,flow cytometry,alkaline phosphatase(ALP)and alizarin red(ARS)staining as well as oil red O staining;(2)To investigate the effect of LINC00704 on the osteo/odontogenic differentiation of SCAP,the expression of LINC00704 was up-or down-regulated by cell transfection.The expression level of osteo/odontogenesis-related genes was detected by qRT-PCR and Western Blot assay,the osteogenic differentiation level was also detected by ALP staining and ARS staining.(3)Bioinformatics analysis and dual luciferase reporter gene assay were used to predict and verify the binding sites of LINC00704 and miR-323a-3p,miR-323a-3p and IGF2BP2;(4)Cell transfection technique and functional experiment were conducted to explore the role of miR-323a-3p and IGF2BP2 in osteo/odontogenic differentiation of SCAP;(5)Cell co-transfection technique and rescue experiment were used to verify the regulatory relationship of LINC00704/miR-323a-3p/IGF2BP2 in osteo/odontogenic differentiation of SCAP.Research results:(1)SCAP,which were isolated and cultured by tissue enzymatic digestion,showed clonelike growth,positively expressed surface markers of mesenchymal stem cells,and could differentiate into osteogenic and adipogenic cells under induction;(2)LINC00704 promoted the osteo/odontogenic differentiation of SCAP;(3)LINC00704 acted as a molecular sponge to adsorb miR-323a-3p to suppress its expression.MiR-323a-3p has a negative regulatory effect on osteo/odontogenic differentiation of SCAP.MiR323a-3p inhibitor can antagonize the inhibitory effect of interfering LINC00704 on osteo/odontogenic differentiation of SCAP;(4)MiR-323a-3p targeted binding IGF2BP2 and negatively regulated its expression.Interference of IGF2BP2 could inhibit osteo/odontogenic differentiation of SCAP and reverse the promoting effect of miR-323a-3p inhibitor on osteo/odontogenic differentiation of SCAP;(5)LINC00704 up-regulated the expression of IGF2BP2 by adsorbing miR-323a-3p,and interfering IGF2BP2 could reverse the promoting effect of LINC00704 on osteo/odontogenic differentiation of SCAP.Conclusion:(1)It is found for the first time that lncRNA LINC00704 promotes osteo/odontogenic differentiation of SCAP;(2)LINC00704 could up-regulate the expression of IGF2BP2 by sponging miR-323a-3p;(3)LINC00704 regulated osteo/odontogenic differentiation of SCAP through miR-323a3p/IGF2BP2 axis. |
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