[1]刘晓静,许佳伟,罗志文,等.耐药骨肉瘤细胞通过细胞外囊泡递送微小RNA-135b-5p/微小RNA-9-5p抑制CDX2诱导化疗敏感细胞干性与耐药[J].陕西医学杂志,2026,(7):867-876，887.[doi:DOI:10.3969/j.issn.1000-7377.2026.07.001]
　LIU Xiaojing,XU Jiawei,LUO Zhiwen,et al.Chemoresistant osteosarcoma cells deliver EV-miR-135b-5p/miR-9-5p toinduce stemness and drug resistance in sensitive cells by targeting CDX2[J].,2026,(7):867-876，887.[doi:DOI:10.3969/j.issn.1000-7377.2026.07.001]
点击复制

耐药骨肉瘤细胞通过细胞外囊泡递送微小RNA-135b-5p/微小RNA-9-5p抑制CDX2诱导化疗敏感细胞干性与耐药

《陕西医学杂志》[ISSN:1000-7377/CN:61-1281/TN]

卷:
期数:
2026年7期
页码:
867-876，887
栏目:
基础研究
出版日期:
2026-07-05

文章信息/Info

Title:
Chemoresistant osteosarcoma cells deliver EV-miR-135b-5p/miR-9-5p toinduce stemness and drug resistance in sensitive cells by targeting CDX2
作者:
刘晓静; 许佳伟; 罗志文; 张弘韬; 张毓健; 闫康; 牛舜; 龙华; 张云飞; 董川
（空军军医大学第二附属医院，陕西 西安 710038）
Author(s):
LIU Xiaojing; XU Jiawei; LUO Zhiwen; ZHANG Hongtao; ZHANG Yujian; YAN Kang; NIU Shun; LONG Hua; ZHANG Yunfei; DONG Chuan
（Second Affiliated Hospital of Air Force Military Medical University,Xi’an 710038，China）
关键词:
骨肉瘤; 化疗耐药; 肿瘤干细胞; 细胞外囊泡; 微小RNA-135b-5p/微小RNA-9-5p; 尾型同源盒转录因子2
Keywords:
Osteosarcoma; Chemotherapy resistance; Cancer stem cells; Extracellular vesicles; miR-135b-5p/miR-9-5p; CDX2
分类号:
R 738.1
DOI:
DOI:10.3969/j.issn.1000-7377.2026.07.001
文献标志码:
A
摘要:
目的：探讨化疗耐药骨肉瘤细胞来源的细胞外囊泡（EV）及其携带的微小RNA-135b-5p/微小RNA-9-5p（microRNA-135b-5p/microRNA-9-5p，miR-135b-5p/miR-9-5p）在调控敏感骨肉瘤细胞干性表型与化疗耐药性中的作用与分子机制。方法：采用浓度梯度递增诱导法构建多柔比星（DOX）和顺铂（CDDP）耐药的骨肉瘤细胞株（SOSP9607）。通过超速离心法提取其条件培养基中的EV（DOX-EV，CDDP-EV），并利用透射电镜、纳米颗粒追踪分析（NTA）及共聚焦显微镜进行鉴定与摄取验证。采用细胞计数试剂盒（CCK-8）检测细胞活力与药物敏感性；蛋白质印迹法（Western blot）和实时荧光定量聚合酶链反应（qPCR）检测相关蛋白及mRNA表达；通过肿瘤成球实验与乙醛脱氢酶活性检测（ALDH）评估细胞干性。利用Target Scan预测并筛选miRNA的靶基因，通过双荧光素酶报告基因实验（Luciferase Assay）进行验证。结果：与敏感细胞来源的EV（CS-EV）比较，DOX-EV或CDDP-EV处理能显著增强敏感细胞在相应化疗药物下的存活率（均P<0.05），并上调其干性相关基因（如SOX2、NANOG）表达、增强肿瘤成球能力及ALDH活性（均P<0.05）。敲低耐药细胞中miRNA加工关键酶DICER1后，其EV促存活与促成球能力被显著削弱（P<0.05）。在敏感细胞中过表达miR-135b-5p或miR-9-5p可模拟上述EV处理表型，并显著降低靶基因尾型同源盒转录因子2（CDX2）的表达（P<0.05）；而抑制这两种miRNA则能逆转EV对CDX2的下调作用（P<0.05）。直接敲低CDX2表达可导致敏感细胞的干性表型增强及化疗耐药性增加（P<0.05）。对临床样本数据的生物信息学分析显示，化疗耐药骨肉瘤组织中干性相关基因集显著富集。结论：化疗耐药骨肉瘤细胞可通过分泌富含miR-135b-5p/miR-9-5p的EV，靶向抑制敏感细胞中CDX2的表达，从而诱导其干性表型并获得化疗耐药性。本研究揭示了EV-miRNA介导的细胞间通讯在骨肉瘤化疗耐药传递中的新机制。
Abstract:
Objective:To investigate the role and molecular mechanism of extracellular vesicle (EV)-encapsulated miR-135b-5p/miR-9-5p,derived from chemoresistant osteosarcoma cells,in regulating the stemness phenotype and chemoresistance of chemosensitive osteosarcoma cells.Methods：Doxorubicin (DOX)-and cisplatin (CDDP)-resistant osteosarcoma cell lines (SOSP9607) were established using a concentration gradient induction method.EVs (DOX-EV,CDDP-EV) were isolated from conditioned media by ultracentrifugation and characterized by transmission electron microscopy,nanoparticle tracking analysis,and confocal microscopy for uptake verification.Cell viability and drug sensitivity were assessed by CCK-8 assay.Protein and mRNA expression levels were detected by Western blot and quantitative real-time PCR (qPCR),respectively.Stemness properties were evaluated via sarcosphere formation assay and ALDH activity measurement.Potential target genes of the miRNAs were predicted using Target Scan and validated by dual-luciferase reporter assay.Results:Treatment with DOX-EV or CDDP-EV significantly enhanced the viability of chemosensitive cells under corresponding drug pressure compared to control EVs (CS-EV) (all P<0.05).Furthermore,this treatment upregulated the expression of stemness-related genes (SOX2,NANOG),increased tumorosphere formation capacity,and elevated ALDH activity (all P<0.05).Knockdown of DICER1,a key enzyme for miRNA biogenesis,in resistant cells attenuated the pro-survival and pro-sphere-forming effects of their derived EVs (P<0.05).Overexpression of miR-135b-5p or miR-9-5p in sensitive cells phenocopied the EV-induced effects and significantly downregulated the expression of the target gene CDX2 (P<0.05).Inhibiting these miRNAs reversed the EV-mediated downregulation of CDX2 (P<0.05).Direct knockdown of CDX2 in sensitive cells enhanced their stemness phenotype and chemoresistance (P<0.05).Bioinformatics analysis of clinical sample data indicated significant enrichment of stemness-related gene sets in chemoresistant osteosarcoma tissues.Conclusion:Chemoresistant osteosarcoma cells can deliver EV-encapsulated miR-135b-5p/miR-9-5p to sensitive cells,which in turn downregulates CDX2 expression.This process promotes the acquisition of a stemness phenotype and confers chemoresistance to the recipient cells.This study reveals a novel mechanism of intercellular communication via EV-miRNAs in the propagation of chemoresistance in osteosarcoma.

参考文献/References:

[1]STRAUSS S J,FREZZA A M,ABECASSIS N,et al.Bone sarcomas：ESMO-EURACAN-GENTURIS-ERN paedcan clinical practice guideline for diagnosis,treatment and follow-up[J].Ann Oncol,2021,32(12)：1520-1536.
[2]FERRARI S,SERRA M.An update on chemotherapy for osteosarcoma[J].Expert Opin Pharmacother,2015,16(18)：2727-2736.
[3]PALMERINI E,TORRICELLI E,CASCINU S,et al.Is there a role for chemotherapy after local relapse in high-grade osteosarcoma?[J].Pediatr Blood Cancer,2019,66(8)：e27792.
[4]BATLLE E,CLEVERS H.Cancer stem cells revisited[J].Nat Med,2017,23(10)：1124-1134.
[5]STEINBICHLER T B,DUDS J,SKVORTSOV S,et al.Therapy resistance mediated by cancer stem cells[J].Semin Cancer Biol,2018,53：156-167.
[6]TORREGGIANI E,RONCUZZI L,PERUT F,et al.Multimodal transfer of MDR by exosomes in human osteosarcoma[J].Int J Oncol,2016,49(1)：189-196.
[7]STEINBICHLER T B,DUDS J,SKVORTSOV S,et al.Therapy resistance mediated by exosomes[J].Mol Cancer,2019,18(1)：58.
[8]KALLURI R,LEBLEU V S.The biology,function,and biomedical applications of exosomes[J].Science,2020,367(6478)：eaau6977.
[9]VAN NIEL G,D’ANGELO G,RAPOSO G.Shedding light on the cell biology of extracellular vesicles[J].Nat Rev Mol Cell Biol,2018,19(4)：213-228.
[10]WANG J,SHI K.Role of tumor-derived exosomes and immune cells in osteosarcoma progression and targeted therapy[J].Front Immunol,2025,16：1658358.
[11]MENNDEZ S T,GALLEGO B,MURILLO D,et al.Cancer stem cells as a source of drug resistance in bone sarcomas[J].J Clin Med,2021,10(12)：2621.
[12]ADHIKARI A S,AGARWAL N,WOOD B M,et al.CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance[J].Cancer Res,2010,70(11)：4602-4612.
[13]GLAVIANO A,LAU H S,CARTER L M,et al.Harnessing the tumor microenvironment：Targeted cancer therapies through modulation of epithelial-mesenchymal transition[J].J Hematol Oncol,2025,18(1)：6.
[14]LIAO Y,CHEN X,XU H,et al.N6-methyladenosine RNA modified BAIAP2L2 facilitates extracellular vesicles-mediated chemoresistance transmission in gastric cancer[J].J Transl Med,2025,23(1)：320.
[15]HE J,WANG L,YU B,et al.Extracellular vesicle-derived microRNAs in renal cell carcinoma：Biological roles and clinical applications[J].Front Cell Dev Biol,2025,13：1694257.
[16]WANG Q,SUN J,JIANG H,et al.Emerging roles of extracellular vesicles in oral and maxillofacial areas[J].Int J Oral Sci,2025,17(1)：11.
[17]YANG Q,LIU J,WU B,et al.Role of extracellular vesicles in osteosarcoma[J].Int J Med Sci,2022,19(8)：1216-1226.
[18]LI Y,LIU F,CAI Q,et al.Invasion and metastasis in cancer：Molecular insights and therapeutic targets[J].Signal Transduct Target Ther,2025,10(1)：57.
[19]LIAO Y,YI Q,HE J,et al.Extracellular vesicles in tumorigenesis,metastasis,chemotherapy resistance and intercellular communication in osteosarcoma[J].Bioengineered,2023,14(1)：113-128.
[20]SAW P E,LIU Q,WONG P P,et al.Cancer stem cell mimicry for immune evasion and therapeutic resistance[J].Cell Stem Cell,2024,31(8)：1101-1112.
[21]LUO Z,LIU M,ZHANG H,et al.Association of circulating miR-125b and survival in patients with osteosarcoma:A single center experience[J].J Bone Oncol,2016,5(4)：167-172.
[22]JIANG M,JIKE Y,LIU K,et al.Exosome-mediated miR-144-3p promotes ferroptosis to inhibit osteosarcoma proliferation,migration,and invasion through regulating ZEB1[J].Mol Cancer,2023,22(1)：113.
[23]XU J F,WANG Y P,ZHANG S J,et al.Exosomes containing differential expression of microRNA and mRNA in osteosarcoma that can predict response to chemotherapy[J].Oncotarget,2017,8(44)：75968-75978.
[24]WANG X,ZHOU Y,DING K.Roles of exosomes in cancer chemotherapy resistance,progression,metastasis and immunity,and their clinical applications (Review)[J].Int J Oncol,2021,59(1)：44.
[25]JIN H,LUO S,WANG Y,et al.MiR-135b stimulates osteosarcoma recurrence and lung metastasis via notch and wnt/β-catenin signaling[J].Mol Ther Nucleic Acids,2017,8：111-122.

相似文献/References:

[1]姚红月,李 岩,陈美雪,等.miR223p靶向负调控eIF4E对儿童骨肉瘤细胞增殖影响的研究[J].陕西医学杂志,2020,49(1):16.
　YAO Hongyue,LI Yan,CHEN Meixue,et al.Effect of miR223p targeting negatively regulated eIF4E on the proliferation of osteosarcoma cells in children[J].,2020,49(7):16.
[2]张 哲,程漠松,庄 鹤,等.骨肉瘤细胞中miR-215-5p和eIF2a靶向关系及临床意义[J].陕西医学杂志,2021,50(7):886.[doi:DOI:10.3969/j.issn.1000-7377.2021.07.031]
　ZHANG Zhe,CHENG Mosong,ZHUANG He,et al.Targeting relationship between miR-215-5p and eIF2a in osteosarcoma cells and its clinical significance[J].,2021,50(7):886.[doi:DOI:10.3969/j.issn.1000-7377.2021.07.031]
[3]张素冰,高 辉,赵滨滨,等.罗哌卡因对骨肉瘤细胞增殖、侵袭及雷帕霉素靶蛋白/缺氧诱导因子1α信号通路的影响[J].陕西医学杂志,2023,52(6):666.[doi:DOI:10.3969/j.issn.1000-7377.2023.06.007]
　ZHANG Subing,GAO Hui,ZHAO Binbin,et al.Effects of ropivacaine on proliferation,invasion and mTOR/HIF-1α signaling pathway of osteosarcoma cells[J].,2023,52(7):666.[doi:DOI:10.3969/j.issn.1000-7377.2023.06.007]
[4]李 程,关贵超,李 兵,等.骨肉瘤组织锌-α2-糖蛋白1表达及其与患者预后关系[J].陕西医学杂志,2024,(12):1709.[doi:DOI:10.3969/j.issn.1000-7377.2024.12.025]
　LI Cheng,GUAN Guichao,LI Bing,et al.Expression of AZGP1 in osteosarcoma and its relationship with prognosis[J].,2024,(7):1709.[doi:DOI:10.3969/j.issn.1000-7377.2024.12.025]
[5]杨玉霞,车姣子,贾利颖,等.鞣花酸通过JAK2/STAT3信号通路介导的巨噬细胞M2型极化抑制骨肉瘤细胞恶性表型及致瘤性实验研究[J].陕西医学杂志,2025,54(9):1177.[doi:DOI:10.3969/j.issn.1000-7377.2025.09.004]
　YANG Yuxia,CHE Jiaozi,JIA Liying,et al.Ellagic acid inhibits malignant phenotype and tumorigenicity of osteosarcoma cells through M2 polarization of macrophages mediated by JAK2/STAT3 signaling pathway[J].,2025,54(7):1177.[doi:DOI:10.3969/j.issn.1000-7377.2025.09.004]

备注/Memo

备注/Memo:
陕西省自然科学基础研究计划一般项目（青年）(2024JC-YBQN-0866);西安市科技计划医学研究一般项目(2024JH-YLYE-0326);唐都医院学科助推计划交叉融合项目(2025JCRH028，2024JCRH006)
更新日期/Last Update: 2026-07-10