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