| Background: Bone defect is mainly caused by bone injury caused by severe impact,trauma,tumor resection,osteoarthritis or secondary surgery.Bone defect repair is mainly treated by bone graft.Bone marrow mesenchymal stem cells(BMMSCs)have been widely used in clinical treatment and experimental research of bone defects in recent years,due to their osteogenic differentiation function.BMMSCs are simple to be harvested,easy to be cultured and have no immune rejection.As the probability of BMMSCs to differentiate into bone is affected by many factors,it is of great clinical significance to study the mechanism of BMMSCs to transform into bone.Objective: In this study,the expression of miRNA-149 was down-regulated by methylation of microRNA-149,or the interaction between lncRNA-h19 and miRNA-149.Thus,the expression of stromal cell-derived factor-1(SDF-1)was up-regulated;the effect of BMMSCs on osteogenic differentiation was observed,and the molecular mechanism of how to down-regulate the expression of miRNA-149,to up-regulate SDF-1 and to stimulate BMMSCs to transform into osteoblast,was explored.Methods: Using targetscan and starbase,SDF-1 gene was found to be the target gene downstream of miR-149,whereas h19 was the upstream regulated lncRNA of miR-149,and the relationship between miR-149 and SDF-1 and lncRNA was verified by double luciferase reporter gene analysis.The expression of H19,miR-149 and SDF-1 in periosteum was detected by immunohistochemistry.The calcium deposition of periosteum was observed by Von Kossa staining.ALP activity of rat periosteum.was detect by PNP colorimetric method.The OCN content of rat periosteum was detected by ELISA.BMMSCs that were isolated from rats,were treated with methyltransferase inhibitor Aza-CdR,SDF-1 neutralizing antibodies,CXCR4 antagonist AMD3100,H19,sh-SDF-1,miR-149 mimic,miR-149 inhibitor;then calcium nodules(Von Kossa staining),mineralized nodules(alizarin red S staining),ALP and OCN were detected.,and CD44 ?CD90 ?CD14 and CD45 were detected by flow cytometry,and the expression of ALP,OCN,RUNX2,OSX were detect by Western blot.Results:(1)The osteogenic differentiation model of rats was successfully established.Compared with sham group,the expression of STRO-1 protein,ALP activity and OCN content in the osteogenic differentiation model group were significantly increased at week 2 and week 4(P< 0.05).BMMSCs were isolated and osteogenic differentiation was induced successfully.After 3 weeks of osteogenic differentiation induced by BMMSCs,the cell morphology changed from spindle to polygon,and calcium salt nodules were seen.Compared with the control group,the ALP activity and OCN content in the bone differentiation induced group were significantly enhanced(P< 0.05).(2)According to bioinformatics analysis,there was a specific binding region between the 3 'UTR of SDF?1 and the miR-149 sequence.Compared with the control group,miR-149 significantly inhibited the luciferase activity of SDF-1-wt 3 '-utr(P<0.05).Compared with the control group,the SDF-1 expression of BMMSCs with a miR-149 mimic was significantly decreased(P<0.05)and significantly increased with miR-149 inhibitor group(P<0.05).Compared with the control group,the ALP activity,OCN content,number of mineralized and calcified nodules,RUNX2 and OSX protein expression of BMMSCs in the miR-149 inhibitor group were significantly increased,while that of the miR-149 mimic group was significantly decreased(P<0.05).(3)Compared with the control group,the methylation level of miR-149 in the bone differentiation induced group was higher,while the methylation level of mir-149 in the 5-Aza-CdR group was lower.miR-149 expression was significantly reduced in the 5-Aza-CdR group on days 7 and 12 compared with the DMSO group(P<0.05).Compared with the bone differentiation induced group,the ALP activity,OCN content,CD44 and CD90 positive rates of the Aza-CdR group were significantly reduced(P<0.05).(4)Compared to the control group,the expression levels of mir-149 in the bone differentiation induction group,the SDF-1 neutralizing antibody group or the AMD3100 group decreased significantly.Compared with bone differentiation induction group,ALP activity,OCN content,exprssion of Nanog,Oct-4 and Sox-2 in SDF-1 neutralizing antibody group or AMD3100 group were decreased(P<0.05).(5)Bioinformatics analysis showed that mir-149 and H19 had partial complementary bases;The expression of mir-149 and H19 in the periosteum of osteogenic differentiated rats was negatively correlated(P<0.05).Compared with anti-IgG,H19 and mir-149 were significantly enriched in the anti-AGO2 group(P<0.05)..Compared with the sham group,the expression of H19 of BMMSCs in the osteogenic differentiation group was significantly increased(P<0.05).Compared with the BMMSCs group treated with sh-h19,the number of calcified and mineralized nodules,ALP activity,OCN content,RUNX2 and OSX protein expression in the H19 overexpression of BMMSCs were significantly increased(P<0.05).(6)Compared with the control group,BMMSCs with H19+ mir-149 mimic showed decreased H19 expression,ALP activity,OCN content,number of calcified and mineralized nodules,and RUNX2 and OSX protein expression,while those with H19+ mir-149 inhibitor increased(P<0.05).Compared with the h19-nc group,sdf-1 expression,ALP activity,OCN content,mineralized nodules,calcified nodules,RUNX2 and OSX expression were significantly increased in the H19 group(P<0.05).Compared with H19+ sh-sdf-1-nc,sdf-1 expression,ALP activity,OCN content,mineralized nodules,calcified nodules,RUNX2 and OSX expression of H19+ sh-sdf-1 were significantly increased and decreased(P<0.05).Conclusion: mRNA-149 can bind to the 3 'UTR of SDF-1 gene and down-regulate the expression of SDF-1 protein of BMMSCs in osteogenic transformation,while the methylation of miRNA-149 gene promoter region down-regulates the expression of miRNA-149 in the cytoplasm,and then up-regulates the expression of SDF-1 and CXCR4 to stimulate the osteogenic differentiation of BMMSCs,increase the formation of calcified nodules and mineralized nodules in BMMSCs,and increase of ALP activity and OCN content.lncRNA-H19 interacts with the miRNA-149 gene to down-regulate the expression of miRNA-149,thereby promoting BMMSCs osteogenic differentiation through the SDF-1 /CXCR4 pathway... |
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