| Objective: CircularRNA(circRNA)is a unique non-coding RNA(nc RNA)with circularstructure.Although many human diseases have been confirmed to be closely related to circRNA,However,whethercircRNA has a potential regulatory function on Bone Marrow Mesenchymal Stem Cells(BMMSCs)osteogenic differentiation needs furtherstudy.In this study,high-throughput RNA sequencing(RNA-seq)was used to detect all circRNA and mRNA differentially expressed in the process of osteogenic differentiation of rat Bone Marrow Mesenchymal Stem Cells(rBMMSCs).The Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment pathways of significantly differentially expressed circRNA and mRNAs were analyzed.The circRNA competitive endogenous RNA(ce RNA)regulatory network was constructed based on the bioassay structure.The accuracy of high-throughput sequencing results was detected by Reverse transcription quantitative polymerase chain reaction(q RT-PCR)and the circRNA-miRNA-mRNA regulatory axis that may regulate the osteogenic differentiation of rBMMSCs was screened.The key signaling pathways and moleculartargets of circRNA driving the osteogenic differentiation of rBMMSCs were identified at the molecularlevel and cellularfunctional level.Finally,the effect of circRNA on bone repairand reconstruction in vivo was verified by constructing rat bone defect model.This study provides a reference basis and approach forthe future application of circRNA in bone tissue engineering.Methods: Part I: All circRNAs and mRNAs differentially expressed in the process of osteogenic differentiation of rBMMSCs were obtained by high-throughput sequencing,and the significantly differentially expressed circrnas and mRNAs were selected according to the specific screening criteria(| fold chang| ≥ 2.0,P < 0.05).The ce RNA regulatory network was constructed by bioinformatics analysis.Then GO and KEGG enrichment analysis were used to explore the differentially expressed circRNAs and mRNAs enrichment analysis items,and analyze the pathways that these enrichment items regulate cell osteogenesis.Finally,the accuracy of circRNAs and mRNAs differentially expressed in the sequencing results was verified by fluorescence quantitative PCR,and the circRNA-miRNA-mRNA regulatory axis with potential regulatory function on rBMMSCs osteogenesis was screened out.Part II: The circRNA＿1809/ Mir-370-3p /Kitlg regulatory pathway that may be related to rBMMSCs osteogenesis was screened by bioinformatics analysis techniques and q RT-PCR.rBMMSCs were transfected with si-circRNA＿1809 and Mir-370-3p overexpression/silencing vectors by RNA interference(RNAi).Fluorescence quantitative PCR,western blot,alizarine red and Von Kossa staining were used to determine the effects of circRNA＿1809 and miR-370-3p on osteogenic differentiation.Luciferase reporting test was used to determine the relationship between miR-370-3p and circRNA＿1809.Finally,knockdown circRNA＿1809 combined with knockdown oroverexpression of miR-370-3p to detected the effect on downstream target gene Kitlg,and the mechanism of affecting osteogenic differentiation of rBMMSCs was analyzed.Part III: The femoral bone defect animal model of SD rats was constructed,the circRNA＿1809 knockdown was combined with rBMMSCs to regraft the bone defect of rats.Through a series of methods such as Hematoxylin and Eosin(HE)staining,X-ray,Micro computed tomography(Micro-CT)and cellularimmunohistochemistry,to evaluate the changes of bone mineral density(BMD),bone volume fraction,osteoblast specific transcription factor(Osterix,OSX),osteopontin(OPN)and otherosteogenic indexes in rat femoral defects.The regulation of circRNA＿1809 on bone repairand reconstruction was verified in vivo.Results: Part I: rBMMSCs were isolated and cultured successfully.Cell surface antigens CD45,CD90,CD105 and CD29 were determined by immunofluorescence staining and flow cytometry.A total of 29 circRNAs and 2453 mRNAs were found to be differentially expressed by high-throughput assay.The 10 most differentially expressed circRNAs were screened out from the differentially expressed circRNAs,and the regulatory network of ce RNA was constructed based on bioinformatics prediction and high-throughput sequencing results.Finally,10 differentially expressed circRNAs and 10 mRNAs were selected to verify the changes in theirexpression levels by q RT-PCR.The results showed that the up-regulated ordown-regulated circRNAs and mRNAs in the sequencing results showed the same trend in PCR results,proving that the sequencing results were accurate and reliable.In addition,q RT-PCR results showed that circRNA＿1809 and Kitlg were the most differentially expressed circRNA and mRNA of rBMMSCs during osteogenic differentiation.Part II: Bioassay results ultimately predicted that circRNA＿1809/ Mir-370-3p /Kitlg axis could potentially regulate osteogenic differentiation of rBMMSCs.q RT-PCR results showed that during the process of osteogenic differentiation of rBMMSCs,the expression level of circRNA＿1809 and Kitlg showed significantly increased,mir-370-3p was decreased.The changes of gene dynamic expression were consistent with the regulatory mechanism of ce RNA.ARS and Von Kossa staining showed that knockdown of circRNA＿1809 could significantly inhibit the osteogenic differentiation of rBMMSCs.q RT-PCR and western blot showed that knockdown circRNA＿1809 significantly reduced the expression of Alkaline phosphatase(ALP)and Bone Morphogenetic protein-2(BMP-2).Knockdown of miR-370-3p significantly promoted osteogenic differentiation of rBMMSCs,but overexpression of miR-370-3p inhibited osteogenic differentiation of rBMMSCs.Dual luciferase reportergene assay demonstrated the binding ability of miR-370-3p to circRNA＿1809.miR-370-3p mimics and miR-370-3p inhibitordown-regulated and up-regulated respectively kitlg expression in rBMMSCs.Si-circRNA＿1809 will also lowerKitlg.Si-circRNA＿1809 + Mir-370-3p inhibitorco-transfection reversed part of the down-regulation of SI-circRNA＿1809 on Kitlg.while Si-circRNA＿1809 + miR-370-3p mimics enhanced the down-regulation of Kitlg.Part III: The femursamples of rats were collected foranalysis at the 4th and 8th weeks aftersurgery.HE and Von Kossa staining showed that the amount of new bone formation in the circRNA＿1809 knockdown group was less than that in the control group,and the speed of bone repairwas also slowerthan that in the control group.Micro-CT and X-ray imaging analysis the rats in the circRNA＿1809 knockdown group had lowerbone mineral density and less new bone tissue in the bone defect area compared with the control group.Immunohistochemical results showed that OSX and OPN of osteogenic indexes in circRNA＿1809 knockdown group were lowerthan those in control group.Conclusions: : 1.29 circRNAs and 2453 mRNAs significantly differentially expressed during the osteogenesis of rBMMSCs.Bioinformatics analysis method was used to predict that a large numberof ce RNA regulatory pathways might be associated with the osteogenesis of rBMMSCs,indicating that circRNA is closely related to the osteogenesis of rBMMSCs.2.During the osteogenesis of rBMMSCs,circRNA＿1809 was significantly overexpressed,while miR-370-3p was underexpressed.In addition,luciferase reports proved that there was a competitive relationship between them,and circRNA＿1809 regulated the osteogenesis of rBMMSCs through the adsorption of miR-370-3p.3.miR-370-3p can bind to downstream target gene Kitlg and regulate the expression of Kitlg gene.Kitlg gene can activate the PI3K/Akt signaling pathway to regulate cell proliferation,apoptosis and osteogenic differentiation.Therefore,circRNA＿1809 promote the expression of Kitlg through the adsorption of miR-370-3p,and then may promote the osteogenic differentiation of rBMMSCs by regulating the PI3K/Akt signaling pathway.4.Knockdown circRNA＿1809 modified rBMMSCs transplanted back into the femoral defect area inhibit the bone repairprocess of SD rats in vivo. |
