中国癌症杂志 ›› 2022, Vol. 32 ›› Issue (9): 827-835.doi: 10.19401/j.cnki.1007-3639.2022.09.010

• 论著 • 上一篇    下一篇

DZNep调控乳腺癌外泌体在肿瘤微环境中的作用

吕叶1(), 英叶霞2, 杨文静1, 厚玉瑾1, 袁春秀1, 曹相玫2()   

  1. 1.宁夏医科大学总医院肿瘤医院肿瘤内科,宁夏 银川 750002
    2.宁夏医科大学基础医学院病理学系,宁夏 银川 750002
  • 收稿日期:2022-01-07 修回日期:2022-06-01 出版日期:2022-09-30 发布日期:2022-10-24
  • 通信作者: 曹相玫
  • 作者简介:吕叶(ORCID: 0000-0002-2561-5090),硕士,副主任医师。
  • 基金资助:
    宁夏自然科学基金资助(2020AAC03384)

The effect of DZNep on tumor microenvironment through regulating exosomes derived from invasive breast carcinoma

LÜ Ye1(), YING Yexia2, YANG Wenjing1, HOU Yujin1, YUAN Chunxiu1, CAO Xiangmei2()   

  1. 1. Department of Oncology, Cancer Hospital, General Hospital of Ningxia Medical University, Yinchuan 750002, Ningxia Autonomous Region, China
    2. Department of Pathology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750002, Ningxia Autonomous Region, China
  • Received:2022-01-07 Revised:2022-06-01 Published:2022-09-30 Online:2022-10-24
  • Contact: CAO Xiangmei

摘要:

背景与目的: 乳腺癌是全球女性发病率最高的恶性肿瘤,患者预后差。肿瘤源性外泌体会改变肿瘤微环境并参与调控肿瘤的发生、发展及转移,这将为肿瘤的诊断和治疗提供新的思路。DZNep能够靶向调控H3K27me3组蛋白甲基转移酶的降解,并特异性地诱导肿瘤细胞凋亡,从而抑制多种肿瘤细胞增殖和迁移。本研究旨在探索DZNep对乳腺癌源性外泌体的影响,并观察其通过调节细胞间连接从而改变乳腺癌细胞上皮-间充质转化(epithelial-mesenchymal transition,EMT)的作用。方法: 应用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和在线分析软件GEPIA2分析EZH2在乳腺癌中的表达,使用肿瘤免疫估计资源(Tumor Immunity Estimation Resources,TIMER)分析EZH2与肿瘤微环境中细胞因子及EMT相关蛋白表达的关系。采用DZNep干预乳腺癌MDA-MB-231细胞,采用差速超速离心法提取外泌体,采用蛋白质印迹法(Western blot)检测CD9、CD63和TSG101的表达情况并鉴定外泌体膜结合蛋白的表达,采用纳米颗粒跟踪分析(nanoparticle tracking analysis,NTA)技术对外泌体的布朗运动进行追踪和分析并结合Stokes-Einstein方程式计算出外泌体颗粒的流体力学直径和浓度,采用透射电镜分析外泌体的大小、形态等,以此来探究DZNep对乳腺癌源性外泌体的改变。利用Western blot分别检测EZH2,细胞间连接蛋白如闭合蛋白(occludin)、闭锁小带蛋白-1(zonula occludens-1,ZO-1)、层粘连蛋白(laminin)、水通道蛋白4(aquaporin 4,AQP4)、连接子蛋白43(connexin 43)、claudin 5和Ⅵ型胶原蛋白(collagen Ⅵ),EMT相关蛋白E-cadherin和vimentin的表达情况,分析DZNep对肿瘤微环境的调节机制。结果: EZH2在癌组织中的表达高于癌旁组织,EZH2高表达与Luminal A型乳腺癌免疫细胞和基质细胞上调均呈正相关,与乳腺癌E-cadherin、N-cadherin、vimentin表达呈正相关,与BRCA-Luminal B型E-cadherin表达呈正相关、N-cadherin表达呈负相关,与BRCA-Luminal A型N-cadherin、vimentin表达呈正相关(P<0.05)。CD9、CD63、TSG101的表达表明成功提取了MDA-MB-231细胞的外泌体,经DZNep干预后,外泌体粒径明显减小且数量下降,细胞连接蛋白occludin、ZO-1、laminin、AQP4及connexin 43表达降低,collagen Ⅵ、封闭蛋白5(claudin 5,CLDN5)表达增加;EMT相关蛋白E-cadherin表达增加,vimentin表达降低。结论: DZNep通过抑制EZH2减少乳腺癌细胞中的外泌体生成,进一步改变肿瘤细胞微环境,从而影响EMT现象。

关键词: DZNep, EZH2, 外泌体, 细胞间连接, 上皮-间充质转化

Abstract:

Background and purpose: Invasive breast carcinoma is the most prevalent malignancy in women worldwide and has a poor prognosis. Tumor xenobiotics change the tumor microenvironment and participate in the regulation of tumor occurrence, development and metastasis, which provide new ideas for the diagnosis and treatment of tumors. DZNep can target and regulate the degradation of H3K27me3 histone methyltransferase and specifically induce tumor cell apoptosis, thereby inhibiting cell proliferation and migration in a variety of tumors. In this study, we investigated the effect of DZNep on invasive breast carcinoma exosomes and observed its effect on epithelial-mesenchymal transition (EMT) of breast cancer cells by regulating intercellular junctions. Methods: EZH2 expression in invasive breast carcinoma was analyzed using the The Cancer Genome Atlas (TCGA) database and the online analysis software GEPIA2, and the Tumor Immunity Estimation Resources (TIMER) was used to analyze the relationship between EZH2 and the expressions of tumor microenvironment cytokines and EMT-related proteins. Intervention of invasive breast carcinoma MDA-MB-231 with DZNep and extraction of exosomes using differential ultracentrifugation were performed. Exosome membrane-bound protein expression was identified by detecting the expressions of CD9, CD63 and TSG101 using Western blot. The Brownian motion of exosomes was tracked and analyzed by nanoparticle tracking analysis (NTA) technique, and the hydrodynamic diameter and concentration of exosome particles were calculated in combination with the Stokes-Einstein equation. Transmission electron microscopy analysis of the size and shape of exosomes was carried out. The above methods were used to explore the changes of exogenous exosomes in breast cancer induced by DZNep. In addition, Western blot was used to detect the expressions of EZH2, intercellular junction proteins such as occludin, zonula occludens-1 (ZO-1), laminin, aquaporin 4 (AQP4), connexin 43, claudin5 and collagen Ⅵ, and EMT-related proteins such as E-cadherin and vimentin, respectively, to analyze the regulatory mechanism of DZNep on the tumor microenvironment.Results: EZH2 expression was higher in cancer tissues than in normal tissues. High EZH2 expression was positively correlated with the upregulation of both invasive breast carcinoma immune cells and stromal cells in Luminal A, positively correlated with E-cadherin, N-cadherin and vimentin expressions in invasive breast carcinoma, positively correlated with E-cadherin expression and negatively correlated with N-cadherin expression in BRCA-Luminal B, and positively correlated with N-cadherin and vimentin expressions in BRCA-Luminal A (P<0.05). The expressions of CD9, CD63 and TSG101 showed that the exosomes of MDA-MB-231 cells were successfully extracted. After DZNep intervention, the particle size of the exosomes was significantly reduced and the number was decreased. The expressions of intercellular junction proteins including occludin, ZO-1, laminin, AQP4 and connexin 43 were decreased, and the expressions of collagen Ⅵ and claudin 5 (CLDN5) were increased. The expression of EMT-related protein E-cadherin was increased, while the expressions of vimentin was decreased. Conclusion: DZNep reduces the generation of exosomes in BRCA by inhibiting EZH2, which further alters the tumor cell microenvironment and thus affects the EMT phenomenon.

Key words: DZNep, EZH2, Exosomes, Intercellular junction, Epithelial-mesenchymal transition

中图分类号: