| Background Ankylosing spondying(AS)was a kind of general immune disease.Inflammation and new bone formation were the main characteristcs of ankylosing spondylitis.Vertebral column surrounding ligaments and discus intervertebralis ossification were the final pathological changes.Axial skeleton emerged fibrous degeneration tetanization,then movement function of joints were lost.If they were not identified and intervened in the early stage,progress of disease could lead to a high disability rate and serious impairment of the quality of life.Until now,The etiology of AS was unclear and pathogenesis of AS might be correlated with genetic,environmental,infective,immunological and other factors.Assessment in Ankylosing Spondylitis international Society(ASAS)put forward,if we wanted to treat AS,with control of inflammation,we must solve the ossification tetanization of the ligaments and synovium tissues at the same time.Mechanisms of inflammation and osteogenesis in AS were not clear and needed further study.14-3-3protein was a protein family which was highly conserved,almost expressed in all eukaryotic cells.It included seven subtypes and interacted with a variety of intracellular proteins to participate in the regulation of biological processes such as cell signaling,cell growth,differentiation,adhesion,and regulation of ion channels.Study of this protein was becoming a hot spot recent years.14-3-3n proteins served as bridges.14-3-3 proteins could interact with more than 200 target proteins in a phosphorylation-dependent and phosphorylation-independent ways.14-3-3 proteins played an important role in many cellular processes,such as cell cycle,cellsignal transduction and apoptosis,had a very wide range of biological functions.More and more studies showed that 14-3-3n protein was closely related to inflammation and osteogenesis.14-3-3n protein was related to the expression of ASK-1,PKC,MAPK,MMP,TNF-?,promoting the occurrence of inflammation.14-3-3n protein could regulate the differentiation and function of osteoblasts,osteoclasts and chondrocytes.In the same time,14-3-3? played a role on absorption,remodeling and repair of bone.In long-term chronic inflammatory process in AS,there must be abnormal expression of osteogenic factor.14-3-3? protein was a new proinflammatory mediator which was investigated to upregulate cytokines and was in association with bone erosion in rheumatoid arthritis.There were no previous reports about influence of 14-3-3? protein on pathogenesis of inflammation and new bone formation in AS,also no anyinvestigations published about associations between 14-3-3? protein and signal pathway of osteogenesis in AS.Our research will verify whether inflammatory cytokines suchas MAPK could stimulate expression of hypoxia-inducible factors(hif-a),vascular endothelial growth factor(vegf),and osteogenesis related molecules including bone morphogenetic such as bone morphogenetic protein-2(BMP-2),osteopontin(OPN),osteocalcin(ost)and Runx detected by QRT-PCR and Western-Blot,in bone marrow mesenchymal stemcells(BMSCs)transfected by sh14-3-3? protein,whether14-3-3? protein could influence expression of osteogenesis related molecules in BMSCs and proliferations of bone cells by transfection or gene silencing technology.Our research will provide new data for clinical treatment.Objective To investigate whether 14-3-3n protein could affects osteogenic differentiation of BMSCs by regulating MAPK signaling pathwayMethods Up-regulation and down-regulation of lentiviral vector transfecting sh14-3-3?protein.14-3-3? protein was overexpressed and low expressed in BMSCs.BMSCs were from experimental animals.m RNA expression and protein content of hif-a,vegf,BMP-2,OPN,ost and Runx2 m RNA in BMSCs were detected at different time point(1d,4d,7d,14 d,21d)by QRT-PCR and Western-Blot.14-3-3? protein and MAPK binding were detected by co-immunoprecipitation method.Expression and protein content of BMP-2 and MAPK m RAN in BMSCs were detected by constructing miRNA142-3P or miRNA142-3P overexpression or inhibition.The effect of 14-3-3?protein on the migration of osteocytes was studied by scratch test.Results1.Construction of lentiviral vector and in vitro transfection of 14-3-3?protein BMSCs(extracted from experimental dogs)identification: Compared with the control group,the expression of 14-3-3? m RNA and protein were significantly inhibited after transfection with sh14-3-3? protein by QRT-PCR and Western-blot.2.Knocked down 14-3-3? protein in BMSCs and cultured in vitro.Expression of hif-a,vegf,BMP-2,OPN,ost and Runx2 m RNA were detected at different time point(1d,4d,7d,14 d,21d)by QRT-PCR.The m RNA expressions of the above moleculars were significantly increased and showed a gradual upward trend.Western-Blot also showed that the expression of hif-a,vegf,BMP-2,OPN,ost and Runx2 were significantly increased and represented a gradual upward trend on day 1,day 4,day 7,day 14 and day 21.3.Knocked down 14-3-3? protein in BMSCs and cultured in vitro.The migration ability of BMSCs was obviously enhanced after 24 h culture by scratch experimental.4.BMSCs were cultured under hypoxic environment,expression of 14-3-3? protein and MAPK were down-regulated(P<0.05),Hif-? and BMP-2 were up-regulated(P<0.05),while 14-3-3? protein decreased with MAPK.Knocked down 14-3-3n protein,MAPK was down-regulated and expression of osteogenic protein BMP-2 were up-regulated..When adding caspase3 inhibitor Ac-DEVD-CHO,knocking down 14-3-3? protein again and MAPK was not cleaved by caspase3 and BMP-2 expression was not up-regulated.5.Western-Blot results showed that the level of 14-3-3? protein and MAPK were down-regulated while the level of BMP-2 was up-regulated compared with control group when miR-142-3P was overexpressed.At the same time,m RNA level of 14-3-3?was down-regulated compared with control group,while m RNA of miR-142-3P was up-regulated compared with control group.To construct a model that inhibited miR-142-3P expression,Western-Blot results showed that 14-3-3? protein and MAPK levels were up-regulated compared with control group,and osteogenic protein BMP-2was down-regulated compared with control group.At the same time,m RNA of 14-3-3?was up-regulated compared with control group,while m RNA of miR-142-3P was down-regulated compared with control group.Conclusion1.14-3-3n protein could significantly inhibit osteogenic differentiation of BMSCs by activating MAPK signaling pathway;2.miRNA142-3P was involved in the inhibition of osteogenic differentiation of BMSCs through the inhibition of 14-3-3? protein. |
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