A Study On The Role Of MIF In Regulating Chondro-osteogenic Differentiation Of Human Cartilage Endplate Stem Cell Under Hpoxic Condition

Background and objectivesDegenerative intervertebral disc is the most important reason of low back pain.The degeneration of cartilage endplate(CEP),characterized by the reduced chondrification and the increased ossification,is considered as one of the initial factors responsible for the degeneration of the whole intervertebral disc.Cartilage endplate stem cell(CESC)with the ability of chondrogenesis and osteogenesis,is responsible for the restoration of CEP.CEP is an avascular and hypoxic tissure,the physiologically hypoxic microenvironment of CEP is destructed in degenerative CEP.Hypoxia plays a key role in regulating differentiation fate of stem cells.No study has focused on the regulatory effect of hypoxia on the differentiation fate of CESC until now.MIF,a downstream target of HIF1 A,plays a important role in regulating the metabolism of both cartilage and bone.However,the role of MIF on the chondro-osteogenic differentiation of stem cells has not been investigated.In this study,we investigated the key role of MIF on the chondro-osteogenic differentiation of CESC.MethodsPart 1: The detection of degeneration/hypoxia of CEP and the isolation of CESCThe CEP of patients with/without lumbar disc degeneration were collected.The expression of HIF1A/COL1/COL2 was detected.CESCs were isolated and then subjected to the detection of characteristics of stem cells.Part 2 : To investigate the regulatory effect of HIF1A/MIF pathway on the chondroosteogenic differentiation of CESC.CESCs were induced in the chondrogenic/osteogenic induction medium under normoxia/hypoxia.The inducer/inhibitor of HIF1 A and up-regulation/down-regulation of MIF expression were used to investigate the regulatory effect of HIF1A/MIF pathway on the chondro-osteogenic differentiation of CESC.Part 3 : To investigate the regulatory mode of MIF on SOX9/RUNX2Luciferase reporter assay and chromatin immunoprecipitation were performed to investigate the transcriptional regulation of MIF on SOX9/RUNX2Part 4: To investigate the change of gene and AS in hypoxia using high-throughput microarrayHTA 2.0 microarray was used to detect the change of gene and AS in CESC under hypoxiaResultsPart 1:1.In degenerated CEP,the expression of HIF1 A and COL2 were reduced and the expression of COL1 was increased2.CESC exhibited characteristics of mesenchymal stem cellPart 2:1.Hypoxia promoted chondrogenesis and inhibited osteogenesis of CESC2.Hypoxia promoted chondrogenesis and inhibited osteogenesis of CESC through HIF1A3.The regulatory effect of hypoxia/HIF1 A could be reversed by the intervention of MIF expressionPart 3:1.MIF entered nucleus under hypoxia in CESC2.Under hypoxia,MIF could bind to the promoter region of SOX9/RUNX2,promoted the transcription activity of SOX9 but inhibited the transcription activity of RUNX2Part 4: The difference of DEG and AS between the samples(hypoxia/normoxia)were subjected to GO analysis and KEGG analysis.Some GO function and signaling pathways were enrichedConclusions1.Degenerated CEP exposed to less hypoxia exhibited less chondrification and more ossification2.Hypoxia promoted chondrogenesis and inhibited osteogenesis of CESC through HIF1A/MIF pathway3.Under hypoxia,MIF could regulate the transcriptional activities of SOX9/RUNX24.The enriched GO function and KEGG signal pathway provided some new research direction of the hypoxia-regulated chondrogenesis/osteogenesis.