| ObjectiveTo study the regulation of BMSCs proliferation and apoptosis by HUCMSC-Exos in DOP rat models.To clarify the differentially expressed miRNA,and further study the mechanism of miR-1263/Mob1/Hippo signaling pathway in the process of regulating DOP.Finally,the DOP rat models were used to further verify the regulatory effect of HUCMSC-Exos on DOP.Methods Part one: Exosomes were extracted from HUCMSCs by ultracentrifugation and HUCMSC-Exos were analyzed and identified by TEM,NTA and Western blot.TEM was used to observe the morphology of HUCMSC-Exos.NTA was used to measure the diameter and concentration of HUCMSC-Exos.Western blot was used to detect the specific surface markers of HUCMSC-Exos.We prepared DOP rat models and extracted BMSCs from them.HUCMSC-Exos was subjected to immunofluorescence tracing analysis in vitro.Further,the proliferation of BMSCs were evaluated by CCK-8 assays and apoptosis of BMSCs were evaluated by Western blot.Part two: Total RNA was extracted from DOP models and DOP+Exos models.The differentially expressed miRNAs were screened by miRNA RNA-seq,and q-PCR was performed to detect the differentially expressed miRNAs.Q-PCR was also performed to verify whether the expression of miRNA-1263 is consistent at the tissue level and the cell level.Bioinformatics analysis was used to predict the binding site of the miRNA-1263 with 3'-UTR of target gene.Further,luciferase reporter assays,RNApull down assays and q-PCR were used to verify the interaction between miRNA-1263 and Mob1.Part three: While preparing DOP rat models,PBS,HUCMSC-Exos,HUCMSC-Exos + miRNA-1263 inhibitor,miRNA-1263 mimics were used to treat DOP rat models,and then DOP rat models were divided into five groups,including normal control group,DOP group,DOP + Exos group,DOP + Exos + inhibitor group and DOP + mimics group.The expression of Mob1 was detected by Western blot and q-PCR to further verify the relationship between miRNA-1263 and Mob1,and the expression of YAP was detected by Western blot and q-PCR used to verify whether there was a regulatory effect on the downstream Hippo signaling pathway.Annexin V-FITC/PI assays were performed to detect the regulatory effects of DOP,HUCMSC-Exos and miRNA-1263 on the BMSCs apoptosis.To further verify the regulatory effect of HUCMSC-Exos and miRNA-1263 on DOP rat models,HE staining,micro-CT scanning and quantitative calculation were used to evaluate the structural changes of tibial trabecular bone.Results Part one: To comprehensively identify the purified exosomes released by HUCMSCs,TEM,Nanosight analysis,and Western blot assays were conducted.From TEM observations,HUCMSC-Exos were evenly scattered and had characteristic exosome morphology,such as a lipid membrane structure and a round 100-mm diameter shape.The Nanosight analysis indicated that the size distribution of these nanoparticles ranged predominantly from 100 to 150 nm,which was consistent with the TEM observations.Western blot showed that HUCMSC-Exos expressed several specific exosomal biomarkers including CD9,CD63,CD81,and Alix.Moreover,HUCMSC-Exos were taken up by BMSCs.The results of CCK-8 analysis indicated that the proliferative capacity of BMSCs from DOP models were significantly suppressed when compared with the normal group,whereas the proliferation rate of BMSCs from DOP models was rescued by HUCMSC-Exos.Cell apoptosis was also investigated using Western blot to measure the expression levels of apoptosis-related proteins,including Bad and cleavedcaspase-3.The BMSCs treated with DOP had notably higher expression levels of Bad and cleaved-caspase-3 compared with those of the normal group,whereas the expression levels were significantly inhibited after HUCMSC-Exos treatment.Part two: To investigate the molecular mechanism of HUCMSC-Exos on cell apoptosis,high-throughput miRNA sequencing was performed to identify exosome-related miRNAs.The results of miRNA-seq showed that 14 miRNAs were upregulated,and 7 miRNAs were downregulated in DOP + Exos group.Q-PCR was performed to investigate the upregulated miRNAs.We focused on miR-1263,which had the highest expression in the DOP + Exos group.Bone tissue q-PCR results also verified that miR-1263 was upregulated in DOP + Exos rats when compared with the DOP rats group.We analyzed the potential target genes of miR-1263 by searching online target databases.Additionally,we predicted the binding sites between miR-1263 and Mob1.The luciferase assay showed the miR-1263 mimics reduced luciferase intensity and the miR-1263 inhibitor promoted luciferase intensity.The results revealed that miR-1263 could interact with Mob1.The RNA-pull down assay showed that the fold enrichment of Mob1 in biotinmiR-1263 group was 4.12-folds higher than that of the control group.Additionally,the expression of Mob1 was upregulated when transfected with miR-1263 inhibitor,and the expression of Mob1 was downregulated when transfected with miR-1263 mimics.All these results verified the inhibitory effect of miRNA-1263 through directly binding to 3'-UTR of Mob1.Part three: Western blot and q-PCR were performed to detect the expressions of Mob1 and YAP.The results showed that when compared with the normal control group,the expressions of Mob1 in the DOP group and the DOP+Exos+inhibitor group were significantly up-regulated and the up-regulated expressions of Mob1 were reversed in the DOP+Exos group and the DOP+mimics group.Additionally,when compared with the normal control group,the expressions of YAP in the DOP group and the DOP+Exos+inhibitor group were significantly down-regulated and the down-regulated expressions of YAP were reversed in the DOP+Exos group and the DOP+mimics group.The Annexin V-FITC assay was used to investigate the antiapoptotic effects of HUCMSC-Exos.The apoptotic BMSC number in the DOP group was significantly higher compared with that of the control group;apoptosis was rescued after exosome treatment.When transfected with miR-1263 mimics in the DOP group,the apoptosis of BMSCs was inhibited compared with the DOP group.Moreover,when transfected with miR-1263 inhibitor in the DOP + Exos group,the apoptosis of BMSCs was significantly promoted compared with the DOP + Exos group.HE staining results indicated that the trabecular structure of the tibia in the normal control group was clear and orderly,while the trabeculae in the DOP group were severely damaged,resulting in increased fractures and significantly larger gaps.The trabecular in DOP+Exos group were not damaged obviously,and the arrangement was relatively orderly.The condition of trabecular in the DOP+Exos+inhibitor group was between the DOP group and the DOP+Exos group.The condition of trabecular bone in DOP + mimics group was similar to that of DOP + Exos group.The results of micro-CT indicated that the trabecular structure of tibia in the normal control group was dense,clear and orderly,while the trabecular in the DOP group was severely damaged and basically disappeared.The condition of the trabecular in the DOP+Exos group was similar to that of the normal control group.In the DOP+Exos+inhibitor group,a large number of trabecular bones were missing and the damage was relatively serious.The trabecular bones in the DOP + mimics group were slightly sparse and damaged.The results of quantitative calculation showed that,when compared with the normal control group,there was a significant decrease in BV/TV,Tb.Th and Tb.N while there was a significant increase in Tb.Sp in the DOP group.When compared with the DOP group,there was a significant increase in BV/TV,Tb.Th and Tb.N while there was a significant decrease in Tb.Sp in the DOP+Exos group.When compared with the DOP+Exos group,there was a significant decrease in BV/TV,Tb.Th and Tb.N while there was a significant increase in Tb.Sp in the DOP+Exos+inhibitor group.Conclusions 1.HUCMSC-Exos can be taken up by BMSCs in vitro.HUCMSC-Exos can regulate DOP by promoting the proliferation of BMSCs and inhibiting the apoptosis of BMSCs.2.The expression of miRNA-1263 is significantly up-regulated in BMSCs treated with HUCMSC-Exos.Additionally,the expression of miRNA-1263 in the cell level is consistent with that in the tissue level.Mi RNA-1263 can regulate DOP by interacting with Mob1.3.Mi RNA-1263 regulates the apoptosis of BMSCs through the Mob1/Hippo signaling pathway.HUCMSC-Exos regulates DOP through miRNA-1263/Mob1/Hippo signaling cascade. |
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