The Regulation Of MiR-23a-3p On Osteogenic Differentiation Of Human Periodontal Stem Cells Through Wnt/β-catenin Pathway

Objective:To study the regulatory effect of miR-23a-3p on osteogenic differentiation of human periodontal ligament stem cells（hPDLSCs）and explore the interaction between miR-23a-3p and Wnt/β-catenin pathway in this process,so as to provide a molecular biological theory for studying osteogenic differentiation of hPDLSCs,orthodontic periodontal tissue remodeling and bone regeneration.Methods:1.Healthy premolars extracted for orthodontic treatment were collected.The primary hPDLSCs were cultured by tissue culture and purified by limited dilution.After passage,the proliferation ability of the 3rd generation hPDLSCs was tested,and the growth curve was drawn with Cell Countering Kit-8（CCK-8）.The cells were identified by flow cytometry,immunohistochemistry,colony forming test,osteogenesis and adipogenesis induction.2.The lentivirus vector containing miR-23a-3p inhibitor was constructed and the optimal Multiplicity of Infection（MOI）value was explored.q RT-PCR was used to detect the inhibitory effect of miR-23a-3p,and CCK-8 was used to detect the effect of lentiviral transfection on the proliferation of of hPDLSCs.3.Setting groups:Blank group,miR-23a-3p inhibitor group and inhibitor NC（inhibitor negative control）group.After transfected with lentiviruses,hPDLSCs were induced with osteogenic differentiation medium for 7 days.The expression of osteogenic genes ALP and RUNX 2 were detected by q RT-PCR and Western Blot to evaluate the osteogenic differentiation.4.72 hours after lentivirus transfection,m RNA and protein expression ofβ-catenin,a key molecule of the Wnt/β-catenin pathway,were detected by q RT-PCR and Western Blot.5.CCK-8 was used to detect the effect of the Wnt/β-catenin pathway blocker DKK1（100 ng/m L）on the proliferative activity of PDLSCs.q RT-PCR and Western Blot were used to detect the m RNA and protein expression ofβ-catenin after addition of DKK1 on culture medium for 72 hours.6.Setting 6 groups in total:Blank group,miR-23a-3p inhibitor group,inhibitor NC group,DKK1,miR-23a-3p inhibitor+DKK1 group,and inhibitor NC+DKK1 group.DKK1 was added to the osteogenic induction medium in specific groups.Seven days after osteogenic differentiation,the m RNA and protein expression changes of osteogenic genes ALP,RUNX2 and Wnt/β-catenin pathway related factorsβ-catenin,p-GSK-3βand Cyclin D-1 were detected by q RT-PCR and Western Blot.Results:1.Primary hPDLSCs were successfully cultivated in vitro.hPDLSCs from the 3^rd-5^thgeneration were selected as study subjects due to their stable morphology and good growth.hPDLSCs grew radially and were spindle-shaped.The cell proliferation curve showed an S-shaped pattern.Cell surface molecular markers and cell cycles detected by flow cytometry were consistent with the characteristics of stem cells.Immunofluorescence staining showed positive expression of vimentin and negative expression of keratin,suggesting that the cells were of mesenchymal origin.In addition,the cultured cells had good cloning,osteogenic and adipogenic differentiation abilities.2.The optimal MOI for lentivirus transfection was 20,which had no effect on the proliferation of hPDLSCs.The expression level of miR-23a-3p in the miR-23a-3p inhibitor group was significantly lower than inhibitor NC group（P<0.05）.3.The m RNA and protein levels of ALP and RUNX2 in the miR-23a-3p inhibitor group were significantly increased than inhibitor NC group after 7 days of osteogenesis induction（P<0.05）.4.The m RNA and protein levels ofβ-catenin in miR-23a-3p inhibitor group were significantly up-regulated than inhibitor NC group after hPDLSCs was transfected with lentivirus for 72 h（P<0.05）.5.DKK1 significantly reduced the m RNA and protein levels ofβ-catenin（P<0.05）without affecting the proliferation of hPDLSCs（P>0.05）.6.Compared with the inhibitor NC group,the m RNA expressions ofβ-catenin and Cyclin D-1 and protein expressions ofβ-catenin,p-GSK-3βand Cyclin D-1 in the miR-23a-3p inhibitor group were significantly increased after 7days of osteogenic induction（P<0.05）,suggesting that inhibition of miR-23a-3p could activate the Wnt/β-catenin pathway during the osteogenic differentiation of hPDLSCs.DKK1 could down-regulate the expression of osteogenic genes and Wnt/β-catenin pathway related factors in all groups（P<0.05）.Although DKK1 inhibited osteogenic differentiation and the activity of Wnt/β-catenin pathway in the miR-23a-3p inhibitor group,expression levels of the osteogenic gene and genes relate to the Wnt/β-catenin pathway remained significantly higher than those in the inhibitor NC+DKK1 group（P<0.05）.Conclusion:1.hPDLSCs were successfully cultured and isolated using tissue culture and limited dilution methods.2.The inhibition of miR-23a-3p by lentivirus transfection showed positive regulation on the osteogenic differentiation of hPDLSCs,accompanied by up-regulation of Wnt/β-catenin pathway activity3.After blocking the Wnt/β-catenin pathway,DKK1 significantly down-regulated the osteogenic differentiation of hPDLSCs in the miR-23a-3p inhibitor group.miR-23a-3p inhibitor positively regulated the osteogenic differentiation of hPDLSCs in vitro by activating the Wnt/β-catenin pathway.