脐带间充质干细胞外泌体携载miR-4465抑制食管癌细胞的侵袭及迁移  

Exosomal miR-4465 by human umbilical cord mesenchymal stem cells suppresses invasion and migration of esophageal cancer cell

作　　者：孙峰[1] 谢玮[2] SUN Feng;XIE Wei(Department of Laboratory Medicine,Nantong Tumor Hospital,Nantong 226361,Jiangsu,China;Department of Laboratory Medicine,Nantong First People's Hospital,The Second Affiliated Hospital of Nantong University,Nan-tong 226001,Jiangsu.China)

机构地区：[1]南通市肿瘤医院(南通大学附属肿瘤医院)肿瘤研究所,南通226361 [2]南通市第一人民医院检验科,南通226001

出　　处：《医学研究生学报》2021年第12期1252-1258,共7页Journal of Medical Postgraduates

基　　金：南通市市级科技计划项目(MSZ18140)。

摘　　要：目的间质干细胞分泌的外泌体(MSC-ex)是MSC旁分泌的活性成分,在多种疾病包括肿瘤的治疗中有着重要的应用文章拟探讨人脐带间充质干细胞(MSC)外泌体携载miK-4465抑制食管癌细胞EC109侵袭、迁移的作用及机制方法分离、培养脐带MSCs并提取MSC-ex,透射电镜、纳米颗粒分析观察外泌体的结构.Western blot检测外泌体标志蛋白CD9和CD81表达。超声法将miRNA mimics NC及miR-4465 mimics导入MSC-ex,获得miRNA mimics NC修饰MSC-ex(NC miR-ex)及miR-4465 mimics修饰MSC-ex(miR-4465-ex)。PBS、MSC-ex、miR-4465-ex分别与ECIO9共培养,采用激光共聚焦显微镜观察MSC-ex在细胞内分布,qRT-PCR检测ECl09的miR-4465水平,Transwell和划痕实验观察miR-4465 mimics转染及miR-4465-ex作用后EC109的迁移及侵袭能力,免疫印迹法检测EC109的上皮-间质化相关蛋白的表达。结果与NC miR-ex组(1.00±0.09)比较,miR-4465-ex 25 nmol/L、miR-4465-ex50nmol/L组EC109细胞中miR-4465表达水平(20_26±2.75、34.71±5.26)显著升高(P<0.01),且具有剂量依赖性。与mimicsNC组(84.67±4.60)相比,miR-4465 mimics25 nmol/L(23.67±4.14)转染组EC109细胞的迁移率明显降低(P<0.01),且miR-4465 mimics50nM(10.27±1.32)转染组EC109细胞的迁移率更加明显(P<0.01):与NC miR-ex组(82.35±4.52)比较,miR-4465-ex组(23.12±2.22)EC109细胞的迁移率明显降低(P<0.01),Westem blot结果表明,与PBS和NC miK-ex组相比,miK-4465-ex作用后EC109细胞M-Cadherin(P<0.05)、p-Smad1/2(P<O.Ol)和Vim-entin(P<0.01)表达下调,E-Cadherin表达上调(P<0.01)。结论MSC-ex作为载体携载高水平miR-4465,可能通过下调TGFp/Smadl/2通路抑制食管癌细胞EC109的侵袭、迁移。Objective Esophageal cancer is a common malignant tumor of upper gastrointestinal tract,lacking of ideal treatments at present.The exosomes secreted by mesenchymal stem cells are the active components secreted by mesenchymal stem cell(MSC).They have important applications in the treatment of tumors.This study is aimed to investigate the effects of exosomal miR-4465 from human umbilical cord MSC on the invasion and migration of esophageal cancer cell line EC 109 and the underlyine;mechanisms.Methods Human umbilical cord MSCs were isolated and cultured,and MSC derived exosomes(MSC-ex)were extracted by ExoQuick-TCTM precipitation method.The morphology and structure of exosomes were observed using transmission electron microscope(TEM)and Nanoparticle Tracking Analysis(NTA).The expressions of exosomal markers,including CD9 and CD81 were detected by Western blot.miRNA mimics NC or miR-4465 was introduced into MSC-Ex by electroporation to produce NC miR-ex or miR-4465-ex.EC 109 cells were cocultured with PBS,NC miR-ex or miR-4465-ex.Then,intracellular distribution of MSC-ex in EC109 cells was performed by immunoflurescence using a laser scanning confocal microscope.Real-time quantitative PCR was used to analyze the expression level of miR-4465 in EC 109 cells.The migration and invasion of EC 109 cells were observed by Transwell and wound-healing assay.Western blot was used to detect the expression of proteins associated with markers related to epithelial-mesenchymal transition such as N-Cadher-in,Vimentin,E-Cadherin and p-Smad1/2 in EC 109 cells.Results Isolated MSC-ex exhibited a sphere-shaped morphology with a diameter of 30-150 nm.Western blot analysis indicated that MSC-ex was positive for exosomal markers CD9 and CD81.Incubated MSC-ex localized in the cytoplasm of EC 109 cells.Compared with PBS and MSC-ex groups,the expression of miR-4465 in esophageal cancer cells was significantly upregulated in miR-4465-ex group.Moreover,overexpression of miR-4465 in MSC-ex enhanced the inhibition effects of MSC-ex on invasion(P

关 键 词：人脐带间充质干细胞 外泌体 食管癌细胞 miR-4465 迁移 侵袭 

分 类 号：R735.1[医药卫生—肿瘤]