| BackgroundMaxillofacial bone defect is one of the most common clinical diseases,which has a serious impact on the physical and mental health and quality of life of patients.Due to their osteogenic potential,bone marrow mesenchymal stem cells(BMSCs)are deemed to be the important endogenous cells for use in bone tissue regeneration among mesenchymal stem cells(MSCs).Enhancing the functions of MSCs is considered to be a potential approach for promoting tissue regeneration.Micro RNAs(miRNA,miR)regulate diverse cellular biological process include cell differentiation,proliferation and apoptosis.Studying the expression of miRNAs during osteogenic differentiation of MSCs and the influence of miRNAs on biological processes in MSCs may provide theoretical basis for bone regeneration and treatment of maxillofacial bone defect.Mi RNA-488 has been reported to mediate cell proliferation,differentiation,migration,signal transduction and homeostasis maintenance.Moreover,the osteogenic differentiation markers osteocalcin(OCN)and alkaline phosphatase(ALP)were up-regulated by miR-488-3p-konckdown in MSCs.Bioinformatics analysis showed that miR-488-3p could bind to the 3?UTR of target gene matrix metalloproteinase-1(MMP-1).MMP-1,a secreted protein,also known as interstitial collagenase,have been reported because it can specifically degrade the proteoglycan,one of the major components of extracellular matrix(ECM),promote bone tissue calcification and participate in periodontal tissue remodeling.Moreover,the expression of MMP-1 m RNA was significantly up-regulated during osteogenic differentiation of BMSCs,indicating that MMP-1may have effects on BMSCs differentiation.In this study,we investigated the effects of miR-488-3p/MMP-1 on BMSCs and its mechanism.Materials and Methods1.Effect and mechanism of miR-488-3p on the osteogenic differentiation of human BMSCsPrimary BMSCs were isolated from fresh cancellous bone of human jaws,cultured and characterizated.To induce osteoblastic differentiation,BMSCs were incubated with mineralization-inducing media(MM)for 7 days.Quantitative real-time reverse transcription-polymerase chain reaction(real-time RT-PCR)was used to detect the expression of miR-488-3p.Using databases targetscan,miRDB and miRsystem,MMP-1 was found to be the downstream target gene of miR-488-3p.Double luciferase reporter gene analysis was used to investigate the relationship between miR-488-3p and MMP-1.Furthermore,miR-488-3p mimics and inhibitor were transfected in BMSCs to obtain miR-488-3p overexpression and knockdown,and the efficiency was confirmed by real-time RT-PCR.The expression of MMP-1was analyzed by Western blotting analyses and real-time RT-PCR.After transfected with miR-488-3p mimics and inhibitor,the osteogenic potency of BMSCs was investigated by the ALP activity assay,ALP denser staining and other methods.To further investigate the mechanism of miR-488-3p on the osteogenic differentiation of BMSCs,lentiviral MMP-1 sh RNA(MMP-1sh)was used to knockdown MMP-1.After selection,the knockdown efficiency was confirmed by Western blotting analyses and real-time RT-PCR.miR-488-3p mimics were co-transfected in BMSCs.The expression of MMP-1 was detected by Western blotting analyses and real-time RT-PCR.The osteogenic potency was evaluated by the ALP activity assay,ALP denser staining and real-time RT-PCR.2.Effect and mechanism of MMP-1 on osteogenic differentiation of human BMSCsLentiviral MMP-1 sh RNA(MMP-1sh)was used to knockdown MMP-1.The cell counting kit-8(CCK8)and scratch assay were used to analyze cell proliferation and migration ability,respectively.The osteogenic potency was investigated by the ALP activity assay,ALP denser staining,alizarin staining and other methods.The optimal concentration 20 ng/m L recombinant human MMP-1 protein(rh MMP-1)was used to stimulate BMSCs,designed by ALP activity assay.The CCK8 and scratch assay were used to analyze cell proliferation and migration ability,respectively.The osteogenic potency was investigated by the ALP denser staining,alizarin staining and other methods.Western blotting analysis was used to detect the jun amino-terminal kinases(JNK)and extracellular regulated protein kinases(ERK)signaling pathways.Results1.miR-488-3p inhibited the osteogenic differentiation of BMSCs via down-regulating the expression of MMP-1The results of flow cytometry showed that the cells obtained from human jaws were negative for CD45 and CD34,and positive for the typical mesenchymal cell surface markers CD44,CD90 and CD105,indicating that the cells have the characteristics of mesenchymal stem cells.The result of real-time RT-PCR showed that miR-488-3p expression was suppressed in BMSCs osteogenesis.Double luciferase reporter gene analysis indicated that MMP-1 was the downstream target gene of miR-488-3p.Moreover,miR-488-3p could suppress the osteogenic differentiation of BMSCs.After lentiviral MMP-1 sh RNA(MMP-1sh)was used to knockdown MMP-1 in BMSCs,miR-488-3p mimics were co-transfected in BMSCs to detect the mechanism of miR-488-3p on BMSCs,and the results suggested that the changes of osteogenic differentiation indexes RUNX2,OSX and OPN m RNA expression,ALP activity and ALP denser staining were similar to those of MMP-1,which indicating that miR-488-3p inhibited the osteogenic differentiation of BMSCs via down-regulating the expression of MMP-1.2.MMP-1 promoted osteogenic differentiation potential of BMSCs via the JNK and ERK pathwayMMP-1 expression was up-regulated in BMSCs osteogenesis.Knockdown of MMP-1 attenuated the migration,proliferation and osteogenic differentiation function of BMSCs and decreased the expression of phosphorylation of JNK and ERK in BMSCs,while rh MMP-1 could enhance migration,proliferation and osteogenic differentiation potential of BMSCs and increased the expression of phosphorylation of JNK and ERK in BMSCs.MMP-1-enhanced osteogenic differentiation and expression of phosphorylation of JNK and ERK in BMSCs were repressed by JNK and ERK inhibitors.ConclusionsmiR-488-3p could decrease the osteogenic differentiation of BMSCs via down-regulating the expression of MMP-1.MMP-1 could accelerate the osteogenic differentiation potentials of BMSCs via the JNK and ERK pathway. |
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