Role And Mechanism Of MiR-125b/NKIRAS2/NF-?B Signaling Pathway In Osteogenic Differentiation Of Human Periodontal Ligament Cells

Objective:Periodontal tissue has the ability of reconstruction and regeneration.The process of orthodontic tooth movement requires the balance of bone absorption and bone formation in periodontal tissue.A large number of studies have shown that periodontal ligament cells?PDLCs? lay an important regulatory role in the process of periodontal tissue self-renewal and periodontal disease tissue repair and regeneration,which is the cytological basis for periodontal defect repair.Therefore,clarifying the regulatory mechanism of bone differentiation of PDLCs will help to further optimize the treatment methods of periodontal tissue regeneration and provide basis for realizing the transformation from basic research to clinical application.MiRNAs participate in a series of important processes in the process of life activities.Many studies have shown that miRNAs can promote or inhibit the differentiation of bone precursor cells and osteoblasts by regulating key transcription factors and osteoblast marker genes in osteoblast signaling pathways.Therefore,to study the changes of important post-transcriptional regulatory molecule miRNAs in the process of periodontal ligament cell osteogenic differentiation and explore the possible regulatory mechanism,which can provide theoretical basis for finding possible miRNAs treatment methods,and can provide research basis for improving clinical application of periodontal ligament cells in repairing tissue defects.MiR-125b is one of the earliest miRNA discovered,and plays a very important role in regulating cell proliferation and differentiation.Studies have found that miR-125b can inhibit the expression level of Samd4 during the osteogenic differentiation of human bone marrow MSCs,thus affecting the osteogenic differentiation process of MSCs.Downregulation of miR-125b expression in MSCs can promote the osteogenic differentiation of MSCs.During the osteogenic differentiation of periodontal ligament stem cells,the expression level of miR-125b also has significant changes.However,it has not been reported whether it participates in and regulates the osteogenic differentiation of human periodontal ligament cells.NF-?B plays a very important role in the process of osteogenesis.In vitro experiments prove that inhibiting NF-kB signal pathway can promote the osteogenic differentiation of MC3T3-E1 cells.Inhibition of NF-?B signal pathway can block the inhibitory effect of TNF-?on Smad signal,thus promoting mineralization of MC3T3-E1.NF-?B inhibits the osteogenic differentiation of mesenchymal stem cells by accelerating the degradation of?-catenin.Phased control of IKK-NF-?B signaling pathway can significantly promote bone growth.Some studies have also shown that inhibiting NF-?B activity can promote osteoblastic differentiation of periodontal ligament cells.NKIRAS2 is a key factor in NF-?B signal transduction pathway,and studies have found that NKIRAS2 can inhibit NF-?B mediated signal transduction pathway.In the previous work,we found that NKIRAS2 is one of the target genes of miRNA-125b after analyzing the target gene prediction software.At the same time,combined with relevant literature,we believe that since NF-?B pathway plays an important role in the osteogenic differentiation of periodontal ligament cells,NKIRAS2 inhibitor of this pathway is the target gene of miRNA-125b,and miRNA-125b plays a very important regulatory role in the osteogenic differentiation of mesenchymal stem cells.Therefore,we speculate whether miRNA-125b can affect the osteogenic differentiation process of periodontal ligament cells through targeted regulation of NKIRAS2.This study intends to use in vitro cell experiments to study the biological effect of miRNA-125b on the osteogenic differentiation of human periodontal ligament cells?hPDLCs?and the molecular mechanism of miRNA-125b regulating the osteogenic differentiation of hPDLCs.An in-depth study of the molecular mechanism of hPDLCs osteogenic differentiation will provide theoretical basis for improving the osteogenic differentiation potential of periodontal ligament cells in clinical practice,and will be helpful to optimize the treatment methods of periodontal tissue regeneration.Methods:1.Using enzyme digestion combined tissue block method cultivates PDLCs in vitro.The CD markers were analyzed by immunofluorescence staining and flow cytometry analysis.Cck8 was used for cell proliferation analysis.Ca²⁺concentration,expression of alkaline phosphatase?ALP?,alizarin red staining,ALP staining,real-time PCR and western blot were tested to investigate the osteoblast differentiation potential of PDLCs.The expression pattern of miR-125b was analyzed by real-time PCR during the osteoblastic process.We obtained the lenticirus of miR-125b overexpression and downexpression.In the process of osteogenic differentiation induced by lv-miR-125b,alizarin red staining,ALP staining,Real-time PCR and western blot were tested to investigate the osteoblast differentiation potential of PDLCs.2.Using shRNA to down-regulate the expression level of NKIRAS2.Alizarin red staining,ALP staining,Real-time PCR and western blot were tested to compare the difference of osteoblast differentiation capacity.The level of phosphorylated I?B and nuclear p65 were analyzed by western blot after NKIRAS2 muted.3.Osteogenic differentiation of PDLCs was induced.Real-time PCR was used to detect the expression changes of NKIRAS2 at different time points.Lentivirus transfected cells to overexpress or inhibit the expression of miR-125b.Real-time PCR was used to detect the expression changes of NKIRAS2.We cotransfected LV-anti-miR-125b and sh-NKIRAS2 into PDLCs cells.After 14d osteogenic differentiation,the expression of osteoblast markers was tested by real-time PCR and western blot.The level of phosphorylated I?B and nuclear p65 were analyzed by western blot to verify miR-125b inhibits PDLCs osteoblastic differentiation by enhancing NF-?B Signaling.Results:1.The morphology of the cells observed under light microscope was spindle-forming fibroid cells,positive for anti-vimentin staining and negative for anti-keratin staining,which proved that the cells were of mesoderm origin.Flow cytometry results showed that CD146 and CD90 were positive,CD45 was negative,and the cells were mesenchymal cells.CCK-8 results showed that the proliferation of periodontal ligament cells was good.Osteogenic differentiation-related experimental results show that periodontal ligament cells have good osteogenic differentiation ability.Osteogenic-related gene expression is significantly increased after osteogenic induction compared with the control group.Alizarin red staining shows that the number of mineralized nodules increases after osteogenic differentiation of cells.After being cultured in osteogenic differentiation media for 1,3,7,14,21days,the expression of miR-125b was down-regulated during the osteoblastic differentiation process.ALP activity and the expression of osteoblast marker gene,such as ALP,OCN,and OPN were decreased in cells transfected with LV-miR-125b.These results indicate that the miR-125b negatively regulates osteogenic differentiation in PDLCs.2.By suppressing NKIRAS2 levels in PDLCs,the PDLCs osteogenesis differentiation decreased.It indicated that the suppression of NKIRAS negatively regulates osteogenic differentiation in PDLCs.The cells transfected with sh-NKIRAS2 were found to express higher level of nuclear p65,phosphorylated I?B and lower level of cytoplasmic p65.It showed NKIRAS2 inhibits NF-?B Signaling.3.After osteogenic differentiation of PDLCs,the expression of NKIRAS2 increased.When miR-125b is overexpressed,the expression of NKIRAS2 decreases,and when miR-125b inhibits expression,the expression of NKIRAS2 increases.After 14d osteogenic differentiation,the extent of osteoblast marker genes increase was far less in PDLCs cells cotransfected LV-anti-miR-125b and sh-NKIRAS2 than that effected by LV-anti-miR-125b transfection alone.Changing the endogenous miR-125b expression by using LV-miR-125b LV-anti-miR-125b,the level of p65 and I?B in PDLCs changed.It showed miR-125b effects on PDLCs osteoblastic differentiation through NF-?B signaling.Conclusions:1.MiR-125b inhibits PDLCs osteoblastic differentiation.2.NKIRAS2 inhibits NF-?B signaling and regulates osteogenic differentiation in PDLCs.3.MiRNA-125b negatively regulate periodontal ligament cells'osteogenic differentiation by target NKIRAS2 through NF-?B pathway.