The Mechanism Of Exosomal MiR-100-5p Inhibiting Osteogenesis And Angiogenesis In Non-traumatic Osteonecrosis Of The Femoral Head

Background:Non-traumatic osteonecrosis of the femoral head(NONFH)is a common orthopedic disease and its early diagnosis and treatment are difficult.To date,the main pathophysiological mechanisms of NONFH are decreased osteogenic differentiation and decreased angiogenesis,but the detailed pathophysiological mechanisms remain unclear.Bone marrow-derived mesenchymal stem cells(BMSCs)and Vessel endothelial cells(VECs),with strong proliferation and differentiation potential,play important roles in the repairment and reconstruction of bone tissue.However,we found that the progression of NONFH could not be reversed even when the causative factors have been removed,and the necrotic bone tissue in patients showed limited osteogenesis and angiogenesis.Therefore,we speculated that it might be the bone tissue in the necrotic area of the patient that released some signals,which affected the self-repairment of the BMSCs and VECs.Exosomes are small vesicles that are involved in cell signaling mainly by paracrine means.Almost all cells in the human body can synthesize and secrete exosomes,and almost all cells receive exosome signal transduction.Therefore,we speculated that the bone tissue in the necrotic area of NONFH patients affected the treatment and progress of this disease by releasing exosomes.MiRNAs have been reported to be involved in the initiation and progression of a variety of orthopedic diseases.Based on this,we explored the role of NONFH-exosomes in the progression of the disease from the human bone tissue,cell and animal.We combined the miRNA sequencing to study the molecular mechanisms of the NONFH-exosomes,so as to provide effective therapeutic targets for the disease.Objective:The purpose of this study was to explore the effects of NONFH exosomes on osteogenic differentiation,angiogenesis and femoral head in rats.To investigate the changes of miRNA expression profile in NONFH exosomes;For the screened miRNAs,their functions were further verified,and the molecular mechanism of NONFH-exosomal pathogenicity was explored through dual-luciferase reporting and RNA interference techniques,so as to provide a new therapeutic target for NONFH.Methods:1.Clinical sample characteristics and extraction and identification of exosomes: The femoral head samples from 30 cases of femoral neck fracture(FNF group as control)and 30 cases of NONFH were collected during hip arthroplasty.Clinical data and radiographs of the patients were collected,and the femoral head samples with no statistically significant differences in age,sex and BMI were screened out.The histological changes of FNF and NONFH samples were observed by H&E staining and immunohistochemistry(IHC).Exosomes were extracted by ultra-high speed centrifugation.Exosomes were identified by Transmission electron microscopy(TEM),Nanoparticle tracking analysis(NTA)and detection of exosome markers.Exosome uptake assay was used to observe exosome uptake of human BMSCs(hBMSCs)and human umbilical vessel endothelial cells(HUVECs).2.Pathogenicity of NONFH in vitro and in vivo: hBMSCs and HUVECs were divided into three groups: PBS group,FNF exosome group,and NONFH exosome group.The treated hBMSCs were cultured in osteogenic induction medium or adipogenic induction medium,and the osteogenic differentiation of hBMSCs was evaluated by alkaline phosphatase staining(ALP)staining and alizarin red S staining,and the adipogenic differentiation of hBMSCs was evaluated by oil red O staining.The treated HUVECs were inoculated in the matrix gel and the angiogenic ability of the HUVECs was assessed using a tubule formation assay.The effect of NONFH exosomes on the migration ability of hBMSCs and HUVECs was evaluated using scratch assay.The effects of NONFH exosomes on the femoral head of rats were investigated by injecting them into rats via a tail vein.The effect of NONFH on osteogenesis and angiogenesis was further investigated by RT-PCR and western blot.3.Screening of differential miRNA and functional verification of differential expression of miR-100-5p : The miRNAs in the NONFH-exosomes of the 3 patients and the FNF exosomes of the 3patients were randomly extracted,and the differential miRNAs were screened by miRNA sequencing,and then verified by RT-PCR.NC,agomiR-100-5p(agonist for miR-100-5p)and antagomiR-100-5p(antagonist for miR-100-5p)were transfected into normal hBMSCs and HUVECs to simulate overexpression of miR-100-5p and under-expression of miR-100-5p.The transfected hBMSCs were subjected to osteogenic induction and adipogenic induction.ALP and alizarin red S staining were used to evaluate osteogenic differentiation,and oil red O staining was used to evaluate adipogenic differentiation.Tube formation assay was used to investigate the angiogenic ability of HUVECs.Western blot was further used to investigate the effects of miR-100-5p on osteogenesis and angiogenesis.4.Mechanisms of miR-100-5p inhibiting osteogenic differentiation and angiogenesis: Target genes of miR-100-5p were predicted using the Target Scan7.2 website,and the targeting relationship was demonstrated by dual luciferase gene reporter assay.HBMSCs and HUVECs were divided into three groups: NC group,si BMPR2 group,and si BMPR2+antagomir-100-5p group.Small interfering RNA(siRNA)of the target gene was constructed to knock down the target gene,and the signaling pathway related to the target gene was explored by "rescue experiment" method(that is,the expression of miR-100-5p was inhibited while the target gene was knocked down).Western blot was further used to investigate the effects of target genes on osteogenesis and angiogenesis.5.The mechanism of antagomir-100-5P in rescuing damage caused by NONFH exosomes: HBMSCs and HUVECs were divided into 4 groups:PBS group,NONFH exosome group,NONFH exosome +NC group(treating with NC and NONFH exosome),NONFH exosomes +antagomiR-100-5p(treating with antagonist of miR-100-5p and NONFH exosomes).The use of ALP staining,alizarin red S staining,oil red O staining,and tubule formation experiments demonstrated that antagomiR-100-5p can rescue the damage caused by NONFH exosomes.The mechanism of antagomiR-100-5p antagonizing NONFH exosomes was further investigated by Western blot.Results:1.Bone tissue in the necrotic area of NONFH patients showed decreased osteogenesis and angiogenesis,and increased adipogenesis,which were characterized by decreased osteogenesis and angiogenesis markers such as Runx2,CD31,FGF2,OPN,and ALP,and increased PPARγ,a marker of adipogenic differentiation.NONFH exosomes and FNF exosomes have been successfully isolated from bone tissue and can be taken up by hBMSCs and HUVECs.2.NONFH exosomes can lead to reduced osteogenic differentiation of hBMSCs: after osteogenic induction,the blue particles in ALP staining decreased,the mineralized nodules in alizarin red S staining decreased,and the osteogenic markers such as OCN,Runx2,Collagen Type 1,OPN and ALP decreased.NONFH exosomes promoted adipogenic differentiation of hBMSCs: protein and gene expression of adipogenic differentiation transcription factor PPARγ were increased,and intracellular lipid droplets were increased after adipogenic induction.NONFH exosomes inhibited the angiogenesis of HUVECs: the angiogenesis markers VEGFA and FGF2 decreased,and the number of intact tubules formed decreased.NONFH exosomes can also cause the thinning and collapse of femoral head trabeculae in rats,which are similar to NONFH.3.MiRNA sequencing and RT-PCR demonstrated that miR-100-5p was upregulated in NONFH exosomes.The overexpression of miR-100-5p inhibited the osteogenic differentiation of hBMSCs: after osteogenic induction,the blue particles observed in ALP staining decreased,the mineralized nodules in alizarin red S staining decreased,and the osteogenic markers such as OCN,Runx2,Collagen Type 1,OPN and ALP decreased.Overexpression of miR-100-5p promoted adipogenic differentiation of hBMSCs: the protein expression of adipogenic differentiation transcription factor PPARγ was increased,and the intracellular lipid droplets were increased after adipogenic induction.Overexpression of miR-100-5p inhibited the angiogenesis of HUVECs: vascular markers VEGFA and FGF2 decreased,and the number of intact tubules formed decreased.4.WB and dual luciferase gene reporting assay proved that BMPR2 was the direct target gene of miR-100-5p.Knocking down BMPR2 could inhibit the osteogenic differentiation of hBMSCs: after osteogenic induction,the blue particles in ALP staining decreased,the mineralized nodules in alizarin red S staining decreased,and the osteogenic markers such as OCN,Runx2,Collagen Type 1,OPN and ALP decreased.Knocking down BMPR2 promoted adipogenic differentiation of hBMSCs:the protein expression of adipogenic differentiation transcription factor PPARγ was increased,and the intracellular lipid droplets were increased after adipogenic induction.Knocking down BMPR2 inhibited angiogenesis in HUVECs: the angiogenesis markers VEGFA and FGF2 decreased,and the number of intact tubules formed decreased.Knocking down BMPR2 inhibits phosphorylation of Smad1/5/9 in hBMSCs and HUVECs.The rescue experiment demonstrated that silencing miR-100-5p could rescue the damage caused by knocking down BMPR2.5.Silencing miR-100-5p can reduce the inhibitory effect of NONFH exosomes on osteogenic differentiation of hBMSCs: after osteogenic induction,the blue particles and the mineralized nodules in ALP staining were increased,and the osteogenic markers such as OCN,Runx2,Collagen Type 1,OPN and ALP were increased.Silencing miR-100-5p reduced the promoting effect of NONFH exosomes on adipogenic differentiation of hBMSCs: the protein expression of adipogenic differentiation transcription factor PPARγ was decreased,and the intracellular lipid droplets decreased after adipogenic induction;Silencing miR-100-5p reduced the inhibitory effect of NONFH exosomes on angiogenesis in HUVECs: vascular markers VEGFA and FGF2 were elevated,and the number of intact tubules formed was increased.Conclusion:1.The expressions of adipogenic markers in necrotic bone tissue were up-regulated,while the expressions of osteogenic markers and angiogenesis markers were down-regulated.FNF-exosomes and NONFH-exosomes were successfully isolated from bone tissue and could be taken up by hBMSCs and HUVECs.2.NONFH exosomes can inhibit osteogenic differentiation and angiogenesis,promote adipogenic differentiation,and lead to NONFH changes in the femoral head of rats.3.NONFH exosomes carry a large number of miR-100-5p,which inhibits the osteogenesis of hBMSCs and the angiogenesis of HUVECs by targeting BMPR2 and inhibiting the BMPR2/Smad1/5/9 pathway.4.Silencing miR-100-5p can reduce the inhibition of BMPR2,activate the BMPR2/Smad1/5/9 pathway,and reduce the inhibitory effect of NONFH-exosomes on osteoblastic differentiation and angiogenesis.