| Objective: By studying the role of miRNA secreted in exosomes of osteosarcoma on OS tumor and angiogenesis,and by analyzing tumor size,weight,volume,and number of blood vessels,study the role and mechanism of tumor angiogenesis effect about miR-181 d in osteosarcoma exosomes.Methods: A data set with the serial number "GS65071E" excavated from the GEO database,which lists in detail the types,names and contents of various miRNAs in the specimens taken from patients with osteosarcoma;through the "R" language calculation method,combined with the "dplyr" and "limma" program packages to calculate the 10 miRNAs with the highest content and related to tumor angiogenesis;purchase human osteosarcoma cell line U2-OS,human vascular endothelial cell line HUVEC,culture U2-OS osteosarcoma cells,and then collect exosomes secreted by human osteosarcoma cells U2-OS by differential centrifugation(OS-Exos),using real-time fluorescent quantitative PCR(qRT-PCR)technology to sort the contents of the above 10 miRNAs in exosomes secreted by osteosarcoma cells,and the highest content is miRNA-181 d.Detect the characteristic proteins on the surface of exosomes by Western Blot(WB),and use Nanoparticle Tracking Analysis(NTA)and High-resolution Transmission Electron Microscope(TEM)to verify that the extracted extracellular particles are exosomes.Obtain miRNA-181 d mimics and inhibitors through virus transfection experiments.Vascular endothelial cells phagocytose exosomes by immunofluorescence experiment to verify that exosomes enter vascular endothelial cells to play a role.The Transwell experiment is used to detect the migration and invasion ability of osteosarcoma cells after different treatments(blank group,exosomal group,miRNA-181 d mimic group,miRNA-181 d inhibitor group).Matrigel vascularization experiment reflects the ability of vascular endothelial cells to form blood vessels after different treatments through the number of vascular junctions and the overall blood vessel length.The chicken embryo angiogenesis experiment is used to detect the number of vascular connections and the length of blood vessels after different treatments(blank group,exosome group,miRNA-181 d mimic group,miRNA-181 d inhibitor group)formed by vascular endothelial cells.The degree of endothelial cell formation.Western blot technology(Western Blot,WB)reflects the expression of angiogenesis-related proteins and related signaling pathway proteins in vitro.The in vivo experiment included nude mice hindlimb tumor formation experiment,4 groups of nude mice with different treatments(blank group,exosomal group,miRNA-181 d mimic group,miRNA-181 d inhibitor group),by measuring the body weight of nude mice and hindlimb tumors The size shows the trend of tumor growth,and at the same time,the expression content of related vascular proteins and signal pathway related proteins in the tumor is reflected by Western blotting;the immunofluorescence staining technique is used to stain the vascular-related proteins to directly reflect the angiogenesis in tumor Protein content.At the same time,the tumor sections were stained by immunohistochemistry,and the proliferation and apoptosis indicators were used to indicate tumor growth and activity in each group.Results: 1.The results of biosynthesis suggest that the blood vessel-related miRNAs in human osteosarcoma specimens include miR-181 d,etc.;2.The qRT-PCR results suggest that among the extracted U2-EXOs,miR-181 d is the highest content among the10 miRNAs.3.Nanoparticle Tracking Analysis(NTA)results show that the extracted extracellular material has a diameter range of 30-120nm;the high-resolution transmission electron microscope(Transmission Electron Microscope,TEM)results suggest that the extracted extracellular material is a double-layered round vesicle.Western Blot(WB)results show that the surface of the extracted extracellular material has exosomal-specific proteins CD63,CD9 and Flotillin-1.However,the surface of tumor cells in the control group did not contain these three kinds of proteins.The above results indicated that the extracted materials were exosomes.4.The results of Transwell experiment showed that the invasion and migration ability of osteosarcoma cells in the miR-181 d mimic treatment group was significantly higher than that of the other three groups,which proved that miR-181 d has the ability to promote the migration and invasion of osteosarcoma cells.5.The results of Matrigel-forming blood vessel experiment and chicken embryo-forming blood vessel experiment showed that after different treatments,the number of vascular junctions and total blood vessel length in the miRNA-181 d mimic treatment group were significantly higher than the other three groups,suggesting that miR-181 d was significant enhance the ability of vascular endothelial cells to form blood vessels.6.The results of in vivo and in vitro WB experiments suggest that after the above four treatments,the vascular-related proteins CD31 and α-SMA,and related pathway proteins PI3K/AKT,MEK/ERK protein levels in the miRNA-181 d mimic treatment group were up-regulated and high in the other three groups,miR-181 d can promote angiogenesis through PI3K/AKT and MEK/ERK signaling pathways.7.The results of immunofluorescence staining experiments show that the extracted exosomes can be absorbed by vascular endothelial cells to play a role and after nude mouse tumors are treated with miR-181 d mimics,the expression of vascular-associated protein CD31 is significantly higher than the other three group.8.The results of the nude mouse tumor formation experiment indicated that the weight of the nude mouse group treated with miR-181 d mimics and the volume and weight of the hindlimb tumor were far higher than the other three groups.Conclusions: Strong angiogenesis was observed in the miRNA-181 d mimic group.miRNA-181 d promotes osteosarcoma angiogenesis and tumour growth through the PI3K/AKT and Mek/Erk signalling pathways. |
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