耐药细胞源性外泌体递送的miR-21-5p通过靶向PDCD4促进骨肉瘤增殖和顺铂耐药  

Effects of cisplatin resistant osteosarcoma cell derived exosomes on proliferation and migration of osteosarcoma cells through delivery of miR-21-5p

作　　者：孙超[1] 孟晨阳 赵佳莉[1] 窦蕊[1] 冯卫[1] 郭世炳[1] SUN Chao;MENG Chen-yang;ZHAO Jia-li;DOU Rui;FENG Wei;GUO Shi-bing(Department of Orthopaedic Surgery,the Second Affiliated Hospital of Inner Mongolia Medical University.Huhhot,Inner Mongolia,010020,China)

机构地区：[1]内蒙古医科大学第二附属医院骨科,呼和浩特010020

出　　处：《中国骨与关节杂志》2025年第2期129-145,共17页Chinese Journal of Bone and Joint

基　　金：国家自然科学基金(81960484);内蒙古自然科学基金(2021BS08009)。

摘　　要：目的通过验证骨肉瘤顺铂耐药细胞分别转染miR-21-5p和程序性细胞死亡蛋白4(PDCD4)后,检测其外泌体源miR-21-5p及其靶基因对骨肉瘤耐药性的调控作用;荷瘤实验验证动物血清外泌体miR-21-5p的变化和肿瘤组织中相关基因的表达,探究骨肉瘤耐药细胞外泌体miR-21-5p与靶基因的相关性及其可能的作用机制。方法(1)通过细胞增殖和迁移实验检测过表达或敲低miR-21-5p载体转染的顺铂耐药细胞分泌的外泌体与骨肉瘤细胞共培养的细胞增殖能力和迁移水平;蛋白质印迹法(WB)和定量逆转录聚合酶反应(qRT-PCR)实验检测PDCD4、多药耐药基因1(MDR1)、微管相关蛋白轻链3(LC3)、自噬相关基因Beclin1(Beclin1)、自噬相关基因5(Atg5)的蛋白和miRNA表达水平。(2)通过TargetScan在线工具筛选与验证骨肉瘤细胞miR-21-5p的靶基因。qRT-PCR验证U2OS及其耐药细胞源外泌体中PDCD4的mRNA差异表达;通过细胞增殖和迁移实验检测过表达或敲低PDCD4载体转染的顺铂耐药细胞中PDCD4对细胞增殖能力和迁移水平的影响;WB和qRT-PCR实验检测PDCD4、MDR1、LC3、Beclin1、Atg5的蛋白和miRNA表达水平。(3)观察各组成瘤裸鼠肿瘤生长情况,分析血清外泌体粒径及CD81、CD63的蛋白表达,WB和qRT-PCR检测裸鼠血清外泌体中miR-21-5p、PDCD4的蛋白和mRNA表达水平。免疫组化检测成瘤裸鼠肿瘤组织中相关基因的表达。结果(1)miR-21-5p过表达或敲低载体转染的顺铂耐药细胞差异表达系统结果表明,与U2OS+Exo组相比,U2OS+miR-21-5p mimics(模拟物)/Exo组细胞增殖率和迁移率均显著增高(P﹤0.0001,P﹤0.01),PDCD4的蛋白和mRNA表达水平均显著降低(分别为P﹤0.05和P﹤0.01),而LC3(LC3Ⅱ/LC3Ⅰ)、MDR1、Beclin1、Atg5的蛋白和mRNA表达水平均显著升高(分别为P﹤0.01和P﹤0.001)。(2)TargetScan在线工具预测的PDCD4是miR-21-5p的潜在靶基因,其mRNA的3’非翻译区(3’UTR)中存在miR-21-5p的结合位点;PDCD4�Objective To verify the effect of miR-21-5p and its target genes on drug resistance of osteosarcoma cells transfected with miR-21-5p and PDCD4,respectively.Tumor-bearing experiments were carried out to verify the changes of serum exosomes miR-21-5p and the expression of related genes in tumor tissues,and to explore the relationship between exosomes miR-21-5p and target genes in drug-resistant osteosarcoma cells and its possible mechanism of action.Methods(1)Proliferation and migration experiments were performed to assess the proliferation and migration levels of osteosarcoma cells by co-culturing osteosarcoma cells with exosomes from cisplatin-resistant cells overexpressing or knocked down with miR-21-5p.Western Blot and qRT-PCR were used to detect the expression of PDCD4,MDR1,LC3,Beclin1,Atg5 protein and miRNA.(2)Target genes of miR-21-5p were screened and validated by TargetScan online tool.The differential expression of PDCD4 mRNA in U2OS and its drug-resistant cell-derived exosomes were detected by qRT-PCR.The effect of PDCD4 on cell proliferation and migration was detected by cell proliferation and migration assay.The protein and miRNA expression levels of PDCD4,MDR1,LC3,Beclin1,Atg5 were detected by Western Blot and qRT-PCR Assay.(3)To observe the growth of tumor in nude mice,analyze the expression of CD81 and CD63 protein in serum exosomes,and detect the expression of miR-21-5p and PDCD4 protein and mRNA in serum exosomes by Western Blot and qRT-PCR.Immunohistochemistry was used to detect the expression of related genes in tumor tissues of nude mice.Results(1)miR-21-5p overexpression or knockdown vector-transfected cisplatin-resistant cell differential expression system results indicated that the cell proliferation rate and migration rate of the U2OS+miR-21-5p mimics/EXO group were significantly increased compared with the U2OS+Exo Group(P<0.0001,P<0.01).The protein and mRNA expression levels of PDCD4 were significantly decreased(P<0.05 and P<0.01,respectively).The protein and mRNA expression levels of LC3

关 键 词：外泌体 骨肉瘤 抗药性 肿瘤 微R-21-5p 程序性细胞死亡蛋白4 

分 类 号：R738.1[医药卫生—肿瘤]