携带miR-196b-5p的骨髓间充质干细胞来源外泌体对结肠癌细胞耐药性的影响  

Effect of exosomes derived from bone marrow mesenchymal stem cells carrying miR-196b-5p on drug resistance of colon cancer cells

作　　者：李晓辉 瞿紫微 卢昕 孟庆彬 陈华涛 任骏 谭成沛 LI Xiaohui;QU Ziwei;LU Xin;MENG Qingbin;CHEN Huatao;REN Jun;TAN Chengpei(Department of Gastrointestinal Surgery,Wuhan First Hospital,Wuhan 430022,Hubei Province,China;Depar tment of General Surgery,Shennongjia Forestry Hospital of Traditional Chinese Medicin,Shennongjia 442000,Hubei Province,China)

机构地区：[1]武汉第一医院胃肠外科,湖北武汉430022 [2]神农架林区中医医院普外科,湖北神农架442000

出　　处：《肿瘤》2020年第10期676-686,共11页Tumor

基　　金：2018年湖北省知识创新专项(自然科学基金)(编号:2018CFC814)。

摘　　要：目的:探讨携带有微RNA(microRNA,miRNA,miR)-196b-5p的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)来源的外泌体(exosomes)对结肠癌细胞耐药性的影响及其机制。方法:分离收集BALB/c小鼠BMSCs并采用FCM法进行鉴定;采用脂质体法将miR-196b-5p-模拟物(miR-196b-5p-mimic)或miR-196b-5p-抑制子(miR-196b-5p-inhibitor)转入BMSCs,从培养液中收集BMSCs分泌的外泌体并予以鉴定(透射电子显微镜下观察外泌体及蛋白质印迹法鉴定外泌体标志物的表达情况)。实时荧光定量PCR法检测外泌体中miR-196b-5p的表达水平。用外泌体处理小鼠结肠癌CT26.WT细胞,并用实时荧光定量PCR法检测细胞中miR-196b-5p的表达水平;随后采用不同质量浓度的5-氟尿嘧啶(5-fluorouracil,5-Fu)(0、1.0、1.5和2.0 mg/mL)处理外泌体处理后的CT26.WT细胞,MTT法检测5-Fu处理24和48 h后对细胞活力的影响,FCM法检测对细胞凋亡率的影响;蛋白质印迹法检测CT26.WT细胞中多药耐药蛋白(multidrug resistance,MDR)、多药耐药相关蛋白(multidrug resistance-associated protein,MRP)、肺耐药相关蛋白(lung resistance-related protein,LRP)以及磷脂酰肌醇-3激酶(phosphoinositide-3 kinase,PI3K)-蛋白激酶B(protein kinase B,PKB,又称AKT)通路相关蛋白的表达水平。结果:成功获得miR-196b-5p高表达(或低表达)的BMSCs分泌的外泌体。随着5-Fu浓度的提高,各组CT26.WT细胞的活力均明显降低,凋亡率增加(P值均<0.01),其中以miR-196b-5p过表达组CT26.WT细胞的活力最低,细胞凋亡率最高。在同一浓度5-Fu干预下,miR-196b-5p过表达组CT26.WT细胞中MDR、MRP、LRP、磷酸化PI3K(phospho-PI3K,p-PI3K)及p-AKT蛋白的表达水平均明显低于Exosomes组(未转入mimic或inhibitor的BMSCs分泌的外泌体)(P值均<0.01),而miR-196b-5p低表达组中各耐药相关蛋白的表达水平均明显高于Exosomes组(P值均<0.01)。结论:携带有高水平miR-196b-5p的BMSCs来源的外泌体可降低结肠癌CT26.WT细胞Objective:To investigate the effect of exosomes derived from bone marrow mesenchymal stem cells(BMSCs)carrying microRNA(miR)-196b-5p on the drug resistance of colon cancer cells and its mechanism.Methods:BMSCs of BALB/c mice were isolated and identified by flow cytometry(FCM)assay.MiR-196b-5p-mimic or miR-196b-5p-inhibitor were transfected into BMSCs by liposome,the exosomes secreted by BMSCs in cell culture medium(CM)were separated.After the exosomes were identified(observed using transmission electron microscopy and detected the expression of exosomes markers by Western blotting),the expression level of miR-196b-5p in the exosomes was detected by real-time fluorescent quantitative-PCR.Colon cancer CT26.WT cells of mice were treated with exosomes,the expression of miR-196b-5p in cells was detected by real-time fluorescent quantitative-PCR.CT26.WT cells were treated with different concentrations 5-fluorouracil(5-Fu)(0,1.0,1.5,2.0 mg/mL),after treatment 24 and 48 h the cell viability was detected by MTT assay,apoptotic rate was detected by FCM method.The expression levels of multidrug resistance(MDR),multidrug resistance-associated protein(MRP),lung resistance-related protein(LRP)and phosphoinositide-3 kinase(PI3K)/protein kinase B(PKB,also known as AKT)pathway related protein were detected by Western blotting.Results:The exosomes of BMSCs with high(or low)expression of miR-196b-5p were successfully obtained.With the increase of 5-Fu concentration,the cell viability of CT26.WT cells were decreased and the cells apoptosis rate were increased in each group.Mimic-exosomes group had the lowest viability and the apoptosis rate was the highest.Under the same concentration of 5-Fu,the expression level of MDR,MRP,LRP,p-PI3K and p-AKT in mimic-exosomes group were significantly lower than those in exosomes group(all P<0.01),while the protein levels in inhibitor-exosomes group were significantly higher than those in exosomes group(all P<0.01).Conclusion:BMSCs-derived exosomes carrying high levels of miR-196b-5p can reduce th

关 键 词：结肠肿瘤 骨髓间充质干细胞 外泌体 结肠癌细胞 耐药性 PI3K/AKT通路 

分 类 号：R737.9[医药卫生—肿瘤]