DZNep调控乳腺癌外泌体在肿瘤微环境中的作用

吕叶; 英叶霞; 杨文静; 厚玉瑾; 袁春秀; 曹相玫

doi:10.19401/j.cnki.1007-3639.2022.09.010

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DZNep调控乳腺癌外泌体在肿瘤微环境中的作用

论著 | 更新时间：2025-12-31

- DZNep调控乳腺癌外泌体在肿瘤微环境中的作用
- The effect of DZNep on tumor microenvironment through regulating exosomes derived from invasive breast carcinoma
- 中国癌症杂志 2022年32卷第9期页码：827-835
- 作者机构：
  
  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
- 作者简介：
  
  [ "吕叶（ORCID: 0000-0002-2561-5090）,硕士,副主任医师。" ]
  曹相玫（ORCID: 0000-0001-5231-4288）,博士,副教授。
- 基金信息：
  
  宁夏自然科学基金资助(2020AAC03384)
- DOI：10.19401/j.cnki.1007-3639.2022.09.010
  中图分类号： R737.9
- 收稿：2022-01-07，
  
  修回：2022-06-01，
  
  纸质出版：2022-09-30
- 稿件说明：
移动端阅览
吕叶, 英叶霞, 杨文静, 等. DZNep调控乳腺癌外泌体在肿瘤微环境中的作用[J]. 中国癌症杂志, 2022,32(9):827-835.

Ye LÜ, , Yexia YING, et al. The effect of DZNep on tumor microenvironment through regulating exosomes derived from invasive breast carcinoma[J]. China Oncology, 2022, 32(9): 827-835.
吕叶, 英叶霞, 杨文静, 等. DZNep调控乳腺癌外泌体在肿瘤微环境中的作用[J]. 中国癌症杂志, 2022,32(9):827-835. DOI： 10.19401/j.cnki.1007-3639.2022.09.010.

Ye LÜ, , Yexia YING, et al. The effect of DZNep on tumor microenvironment through regulating exosomes derived from invasive breast carcinoma[J]. China Oncology, 2022, 32(9): 827-835. DOI： 10.19401/j.cnki.1007-3639.2022.09.010.

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

#x02165;型胶原蛋白（collagen

#x02165;）

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（

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

#x02165;、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。

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

#x02165;

and EMT-related proteins such as E-cadherin and vimentin

respectively

to analyze the regulatory mechanism o

f 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 (

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

#x02165; 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.

关键词

Keywords

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