阿霉素耐药细胞外泌体通过传递耐药性影响骨肉瘤细胞增殖和迁移的实验研究

Effects of adriamycin resistance cell-derived exosomes on the proliferation and migration of osteosarcoma cells through drug resistance transmission

目的探讨骨肉瘤阿霉素耐药细胞外泌体与MDR1、miRNAs的相关性及作用机制。方法选取骨肉瘤MG63和U2OS细胞株构建阿霉素耐药株,通过MTT法检测耐药和敏感株的50%抑制浓度(half maximal inhibitory concentration,IC50),于15~120 min中间隔15 min观察一次荧光染色检测荧光强度的变化,RT-PCR和Western Blot检测MDR1的mRNA和P-gp蛋白表达水平等验证骨肉瘤细胞耐药性;通过粒径分析及Western Bolt检测鉴定外泌体。观察PKH26标记的阿霉素耐药细胞外泌体的胞吞作用,并采用MTT法和细胞划痕实验检测阿霉素耐药细胞外泌体与骨肉瘤细胞共培养后细胞的增殖水平、迁移能力及耐药性的变化。通过骨肉瘤源性外泌体测序及生物信息分析、RT-PCR验证miRNAs在骨肉瘤敏感和耐药细胞中的mRNA差异表达水平。观察MG63和U2OS中正常骨肉瘤细胞组与耐药细胞组、耐药细胞+正常外泌体组、耐药细胞+耐药外泌体组成瘤裸鼠肿瘤生长情况,血清外泌体鉴定及其miR-21-5p的mRNA表达水平,RT-PCR、Western Blot和免疫组化检测肿瘤组织中MDR1的mRNA和miR-21-5p蛋白的表达水平。结果MG63和U2OS中阿霉素耐药株IC50(11.81、9.33μg/ml)高于正常株(2.21、0.93μg/ml),荧光强度减弱速度高于正常株,MDR1的mRNA和P-gp的蛋白表达水平(2.15±0.10、2.127±0.12;0.92±0.11、0.73±0.10)均高于正常株(1.12±0.16,1.02±0.11;0.46±0.03、0.30±0.04),差异均有统计学意义(P<0.05)。耐药外泌体分别与正常株共培养时可通过胞吞作用进入骨肉瘤细胞内集中分布于细胞质。骨肉瘤细胞与耐药外泌体共培养,分别以2、4、6、8μg/ml的阿霉素浓度处理后,耐药组增殖水平显著上升,耐药组迁移能力(54.20±9.32,19.24±2.88;76.40±5.41,30.26±4.87)较正常组(35.947±3.922,6.72±3.546;51.217±5.546,19.31±1.930)均高,差异有统计学意义(P<0.05)。选择外泌体测序和生信分析中10种上调明显的miRNAs,进行RT-PCR验证其在正常和耐药株中的mRNA表达差异性,其中耐药株miR-372-3p、miR-21-5p、miR-155-5p表达水平均显著升(MG63:5.89±0.26 vs.0.99±0.06;U2OS:1.05±0.07 vs.8.80±0.93),差异有统计学意义(P<0.05)。MG63和U2OS中正常细胞组与耐药细胞组比较、正常外泌体组与耐药外泌体组比较,后两者裸鼠肿瘤体积呈现不同程度的增速;终末肿瘤重量不同程度地增加。血清外泌体中耐药组miR-21-5p的mRNA表达水平(1.13±0.12、1.14±0.12;0.90±0.07,0.93±0.04)均较正常组(0.86±0.07、0.86±0.05;0.71±0.05,0.75±0.03)增高,差异均有统计学意义(P<0.05)。结论骨肉瘤耐药细胞外泌体通过细胞间传递MDR1基因、miRNAs使细胞的增殖水平和迁移能力增强;耐药细胞及其成瘤裸鼠血清和肿瘤组织中MDR1基因、miR-21-5p表达上调,提示其可能参与调节骨肉瘤MG63、U2OS的耐药过程。

Objective To explore the relationship and underlying mechanism between exosomes derived from doxorubicin-resistant osteosarcoma cells and MDR1 and miRNAs.Methods MG63 and U2OS cell lines were selected to construct doxorubicin-resistant strains,and the 50%inhibitory concentration(half maximal inhibitory concentration,IC50)of drug-resistant and sensitive strains was detected by MTT,and fluorescence staining was performed at intervals of 15 min between 15 and 120 min to detect the change of fluorescence intensity.RT-PCR and Western Blot were used to detect the expression levels of MDR1 P-gp to verify the drug resistance of osteosarcoma cells.Exosomes were identified by particle size analysis and Western Bolt detection.The endocytosis of PKH26-labeled exosomes from doxorubicin-resistant cells was observed,and the proliferation level and migration of exosomes from doxorubicin-resistant cells co-cultured with osteosarcoma cells were detected by MTT assay and cell scratch assay.The differential expression levels of miRNAs in osteosarcoma-sensitive and drug-resistant cells were verified by sequencing and bioinformatics analysis and RT-PCR assay.Tumor growth,serum exosome identification and mRNA expression level of miR-21-5p in tumor-bearing nude mice between normal osteosarcoma cell group and drug-resistant group,drug-resistant+normal exosome group,drug-resistant+drug-resistant+drug-resistant exosome group were observed.MDR1 expression level in tumor tissue was detected by RT-PCR,Western Blot and immunohistochemistry.Results The IC50 of two adriamycin resistant strains were 2.21 vs.11.81μg/ml and 0.93 vs.11.81μg/ml,respectively,and the fluorescence intensity decreased faster than that of normal strains.The relative mRNA expression levels of MDR1 in two cell lines were normal 1.12±0.16,1.02±0.11 and drug-resistant 2.15±0.10,2.127±0.12,respectively.The relative protein expression of P-gp was normal 0.92±0.11,0.73±0.10 and drug-resistant 0.46±0.03,0.30±0.04,the differences were statistically significant(P<0.05).Drug-resistant exosomes can enter osteosarcoma cells through endocytosis and concentrate in the cytoplasm when co-cultured with normal strains.Osteosarcoma cells were co-cultured with drug-resistant exosomes at 2,4,6,and 8μg/ml adriamycin,respectively.Compared with normal group,the proliferation level in drug-resistant group was significantly increased.Compared with the normal cell group 35.95±3.92,6.72±3.55 and the normal exosome group 51.22±5.55,19.31±1.93,the drug-resistant cell group 54.20±9.32,19.24±2.88 and drug-resistant exosome group 76.40±5.41,30.26±4.87,all had significantly higher cell mobility,the difference was statistically significant(P<0.05).Exosome sequencing and biogenic analysis of 10 highly upregated miRNAs to validate mRNA expression differences between normal and drug-resistant strains by RT-PCR,showing a significant increase in miR-21-5p expression level of drug-resistant strains(5.89±0.26 vs.0.99±0.06;1.05±0.07 vs.8.80±0.93,P<0.05),the difference was statistically significant(P<0.05).In MG63 and U2OS,the normal cell group and drug-resistant cell group,and the normal exosome group and drug-resistant exosome group were compared,the tumor volume and the terminal tumor weight of nude mice were increased to varying degrees.MRNA relative expression levels of miR-21-5p in serum exosomes of nude mice after drug intervention were 0.86±0.07 and 0.86±0.05 in normal cell group,respectively.The values were 1.13±0.12,1.14±0.12 in drug-resistant cell group,0.71±0.05,0.75±0.03 in normal exosome group,and 0.90±0.07,0.93±0.04 in drug-resistant exosome group.Compared with normal and drug-resistant strains,the expression levels of normal and drug-resistant exosome groups were increased,with statistical significance(P<0.05).Conclusion The exosomes of drug-resistant cells in osteosarcoma could enhance the proliferation level and migration ability of cells through intercellular transfer of MDR1 and miRNAs.The expression of MDR1 and miR-21-5p in drug-resistant cells and tumor-forming nude mouse serum and tumor tissues were up-regulated which suggested that it might be involved in regulating the drug resistance process of osteosarcoma.