| Objective: MG63 and U2 OS cell lines were screened from 5 osteosarcoma(OS)cell lines for this study.OS cell exosomes were extracted and identified.OS drug-resistant cell lines were constructed to clarify the role of exosomes in intercellular transmission of cisplatin and doxorubicin resistant in OS cells.To investigate the effect of OS-resistant cell-derived exosome on cisplatin and adriamycin resistance and cell proliferation and migration ability.The differences in the expression of resistance-related mi RNAs in OS cells and their exosomes were analyzed.The relationship between exosome and mi RNAs in OS cells and resistance to cisplatin and adriamycin and its possible mechanism was explored.Methods: 1)Osteosarcoma cell lines MG63,U2 OS were screened out: The supernatant exosomes of samples SAOS2,143 B,MG63,U2 OS,HOS and h FOB1.19 cells were analyzed using a nanoparticle size potentiometer.The samples were analyzed by microspectrophotometer to detect the concentration of exosomal RNA in the supernatant of each group of cells;2)Isolation and identification of exosomes: The exosomes of human osteosarcoma cells MG63 and U2 OS were extracted by kits and ultracentrifugation,and their morphology and size were observed by transmission electron microscopy,and Western blotting(Western Blot,WB)Technology to detect the expression levels of exosome transmembrane proteins CD63 and CD81;3)Establishment of osteosarcoma-resistant cell lines: The drug resistance of OS cells was verified by MTT assay and rhodamine 123 staining.After the establishment of drug-resistant strains,OS cell lines were extracted and identified as cis-platinum(CDDP)-resistant cells MG63/CDDP,U2OS/CDDP and adriamycin(adriamycin,ADM)cell MG63/ADM,U2OS/ADM exosomes,the method is the same as above;4)The effect of exosomes on the drug resistance of OS cells MG63 and U2OS: Whether Exo/CDDP and Exo/ADM labeled by PKH26 could enter MG63 and U2 OS cells through endocytosis was observed under inverted fluorescence microscope.MTT assay and scratch assay were used to detect the proliferation rate and migration ability of MG63 and U2 OS cells co-cultured with Exo/CDDP,Exo/ADM.5)Screening of drug resistance-related mi RNA molecules: The m RNA expression levels of 10 mi RNAs in MG63/U2 OS and MG63/U2 OS drug-resistant cells were detected by OS-derived exosome sequencing,bioinformatics analysis,and q RT-PCR.Results: 1).Exosomes of human h FOB1.19,5 OS cell lines and human OS cisplatin-resistant MG63/CDDP,U2OS/CDDP and doxorubicin-resistant MG63/ADM,U2OS/ADM could be obtained.The exosomes of SAOS2,143 B,MG63,U2 OS,HOS and h FOB1.19 cell were analyzed by using a nanoparticle size potentiometer.The obtained average particle size and main peak were all within the particle size range of exosomes,and the particle distribution coefficient was 0.09 to 0.8,which proves that the collected exosomes have moderate dispersion,and the result has a high degree of confidence.The samples were analyzed by microspectrophotometer,and the concentrations of exosomal RNA in the supernatant of SAOS2,143 B,MG63,U2 OS,HOS and h FOB1.19 cells were: 7.5 ng/μl,6.7 ng/μl,8.3 ng/μl,9.2 ng/μl,8.9 ng/μl and7.0 ng/μl,respectively.Under the transmission electron microscope,the exosomes were all round or oval vesicle structures which showed a typical "cup-shaped" structure.The size is 40-130 nm.The results of WB showed that the exosomal transmembrane proteins CD63 and CD81 were expressed at high levels.2)MTT assay showed that compared with OS-sensitive cell lines,the IC50 of drug-resistant strains against CDDP and ADM was significantly improved.The fluorescence observation results of rhodamine 123 staining showed that the fluorescence intensity of OS drug-resistant cells decreased significantly faster than that of sensitive cell lines over time.The expression of MDR1 and P-gp in OS-resistant cell lines were significantly higher than those in OS-sensitive cell lines(p < 0.0001).When Exo/CDDP and Exo/ADM were co-cultured with MG63 and U2 OS cells respectively,they could enter MG63 and U2 OS cells through endocytosis and concentrated in the cytoplasm.MG63 and U2 OS cells were co-cultured with Exo/CDDP and Exo/ADM,and treated with different concentrations of drugs,the proliferation level and migration ability of the cells in each group were significantly increased compared with the OS-sensitive group,among which the drug-resistant Exo group was the most significant,the difference was significant(P<0.0001).3)U2OS exosome was sequenced and bioinformatics analyzed,a total of301 differentially expressed mi RNAs were found by sequencing,including 184up-regulated mi RNAs and 117 down-regulated mi RNAs.The m RNA expression levels of 10 mi RNAs in MG63/U2 OS and MG63/U2 OS resistant strains were detected by QRT-PCR.Compared with normal MG63 cells,the m RNA expression levels of mi R-372-3p,mi R-21-5p and mi R-155-5p in MG63 drug-resistant strains(CDDP,ADM and CDDP+ADM)were significantly increased.Compared with normal U2 OS cells,m RNA expression levels of mi R-372-3p,mi R-21-5p,mi R-155-5p,mi R-138-1-3,mi R-1323 and mi R-130-3p in U2 OS resistant cells were significantly increased.(P <0.0001).Conclusion: 1)Exo can be secreted by MG63 and U2 OS cells including their CDDP and ADM resistant cells.2)Exo of OS drug-resistant cells can be transmited into OS cells and concentrate in the cytoplasm so that exo could enhance the proliferation and migration ability of cells;3)Compared with MG63 and U2 OS cells,mi R-372-3p,mi R-21-5p,and mi R-155-5p were significantly increased in drug-resistant(CDDP,ADM)cells which suggested the generation of drug resistance.These mi RNAs are involved in regulating the cisplatin/doxorubicin resistance of osteosarcoma cells(MG63/U2OS). |
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