The Mechanism Research Of RUNX3 Regulating Hsa_Circ₀005752 To Promote Osteogenic Differentiation Of ADSCs

Background and purpose:Osteoporosis is the crucial cause of osteoporotic fractures,related orthopedic diseases,and even death,affecting the health and quality of life of hundreds of millions of people worldwide.Adipose-derived mesenchymal stem cells(ADSCs)are multilineage potential.Compared with other tissue-derived mesenchymal stem cells,ADSCs have many superior properties and are an ideal source of seed cells in regenerative medicine.Multiple factors regulate the osteogenic differentiation of ADSCs,osteogenesis-related signaling pathways,and epigenetic modification.Circ RNAs are essential epigenetic regulators for osteogenic differentiation of ADSCs.However,the circRNA expression profile of ADSCs osteogenic differentiation is unexplored.And the specific mechanism of how circRNAs regulate the osteogenic differentiation of ADSCs is still unclear.Transcription factors can regulate the expression of circRNAs.This project aims to clarify the specific molecular mechanism of circr Na-mediated osteogenic differentiation of ADSCs,and to clarify that transcription factors regulate the expression of circRNA and then regulate the osteogenic differentiation of ADSCs,so as to provide a new modification strategy for the application of ADSCs in osteoporotic fractures and orthopedic diseasesMethod: Human adipose mesenchymal stem cells(h ADSCs)were obtained for osteogenic induction differentiation,and the expression of circRNA was detected,the differentially expressed circRNA was screened,and the target circRNA was finally obtained.The mechanism of circRNA regulating h ADSCs osteogenic differentiation was systematically studied,and the circRNA was obtained by bioinformatics analysis.The targeted binding relationship between circRNA and miRNA,and miRNA-target genes was detected by luciferase assay.Meanwhile,its effect on h ADSCs osteogenic differentiation was detected to reveal its regulatory mechanism,and transcription factor RUNX3 and circRNA were further detected.Resutls: To analyze circRNAs that regulate osteogenic differentiation,we used the h ADSCs after osteogenic differentiation for14 days.And then,we found that multiple circRNAs were differentially expressed,among which hsa＿circ＿0005752 showed a significantly up-regulated expression trend.We then explored the regulatory role of hsa＿circ＿0005752 in osteogenic differentiation.According to the ceRNA theory,circRNAs usually share the same miRNA as m RNA.Therefore,we predicted and constructed the hsa＿circ＿0005752/miR-496/MDM2 network.Then experiments were performed to explore the function of hsa＿circ＿0005752 in osteogenic differentiation and its interaction with miR-496 and MDM2.Through multiple experiments,we found that miR-496 could target hsa＿circ＿0005752 and MDM2.Meanwhile,we detected MDM2 in h ADSCs overexpressing miR-496 and found that miR-496 significantly reduced MDM2 expression but could be rescued by hsa＿circ＿0005752 overexpression.We also found that overexpression of miR-496 inhibited the osteogenic differentiation of h ADSCs,and overexpression of hsa＿circ＿0005752 could rescue the miR-496-mediated inhibition of osteogenic differentiation of h ADSCs.In further experiments,we explored the mechanism responsible for the increased expression of hsa＿circ＿0005752.We revealed the mechanism by which RUNX3 binds to the LPAR1 promoter and regulates the expression of hsa＿circ＿0005752,thereby affecting the osteogenic differentiation of h ADSCs.Chromatin immunoprecipitation(Ch IP)and luciferase reporter assays confirmed that RUNX3 could target the LPAR1-binding promoter.Further q PCR results showed that overexpression of RUNX3 could up-regulate the level of hsa＿circ＿0005752.At the same time,we observed that the levels of MDM2 and osteogenesis-related factors(RUNX2,Osx,ALP,OCN)were up-regulated.In contrast,the level of p53 was down-regulated,while the level of LPAR1 had no significant change.Conclusion: In conclusion,our research elucidates the molecular mechanism of hsa＿circ＿0005752 regulating the osteogenic differentiation of h ADSCs.We believe that hsa＿circ＿0005752 and MDM2 m RNA jointly targeting bind to miR-496 and regulate the osteogenic differentiation of h ADSCs through the hsa＿circ＿0005752/miR-496/MDM2-p53 ceRNA network.At the same time,the expression of hsa＿circ＿0005752 is targeted by RUNX3,thereby affecting the osteogenic differentiation of h ADSCs.Our findings provide a gene modification strategy to enhance osteogenic differentiation of h ADSCs and offer an essential cell resource for the development of bone tissue engineering.