Department of Biology, Faculty of Science, Anadolu University, 26400, Eskişehir, Turkey.
Department of Biological Sciences, Faculty of Science and Letters, Middle East Technical University, 06800, Ankara, Turkey.
Cell Oncol (Dordr). 2018 Aug;41(4):439-453. doi: 10.1007/s13402-018-0384-6. Epub 2018 Jun 1.
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Complete epithelial to mesenchymal transition (EMT) has long been considered as a crucial step for metastasis initiation. It has, however, become apparent that many carcinoma cells can metastasize without complete loss of epithelial traits or with incomplete gain of mesenchymal traits, i.e., partial EMT. Here, we aimed to determine the similarities and differences between complete and partial EMT through over-expression of the EMT-associated transcription factor Slug in different HCC-derived cell lines.
Slug over-expressing HCC-derived HepG2 and Huh7 cells were assessed for their EMT, chemo-resistance and stemness features using Western blotting, qRT-PCR, neutral red uptake, doxorubicin accumulation and scratch wound healing assays. We also collected conditioned media from Slug over-expressing HCC cells and analyzed its exosomal protein content for the presence of chemo-resistance and partial EMT markers using MALDI-TOF/TOF and ELISA assays, respectively.
We found that Slug over-expression resulted in the induction of both complete and partial EMT in the different HCC-derived cell lines tested. Complete EMT was characterized by downregulation of E-cadherin and upregulation of ZEB2. Partial EMT was characterized by upregulation of E-cadherin and downregulation of vimentin and ZEB2. Interestingly, we found that Slug induced chemo-resistance through downregulation of the ATP binding cassette (ABC) transporter ABCB1 and upregulation of the ABC transporter ABCG2, as well as through expression of CD133, a stemness marker that exhibited a similar expression pattern in cells with either a complete or a partial EMT phenotype. In addition, we found that Slug-mediated partial EMT was associated with enhanced exosomal secretion of post-translationally modified fibronectin 1 (FN1), collagen type II alpha 1 (COL2A1) and native fibrinogen gamma chain (FGG).
From our data we conclude that the exosomal proteins identified may be considered as potential non-invasive biomarkers for chemo-resistance and partial EMT in HCC.
肝细胞癌（HCC）是全球癌症相关死亡的第二大主要原因。完全上皮-间充质转化（EMT）长期以来被认为是转移起始的关键步骤。然而，许多癌细胞在没有完全丧失上皮特征或不完全获得间充质特征的情况下也可以转移，即部分 EMT。在这里，我们旨在通过在不同的 HCC 衍生细胞系中过表达 EMT 相关转录因子 Slug 来确定完全 EMT 和部分 EMT 之间的异同。
使用 Western blot、qRT-PCR、中性红摄取、阿霉素积累和划痕愈合试验评估过表达 EMT 相关转录因子 Slug 的 HCC 衍生 HepG2 和 Huh7 细胞的 EMT、化疗耐药和干性特征。我们还从过表达 Slug 的 HCC 细胞中收集条件培养基，并使用 MALDI-TOF/TOF 和 ELISA 分析其外泌体蛋白中存在的化疗耐药和部分 EMT 标志物。
我们发现 Slug 的过表达导致了不同 HCC 衍生细胞系中完全和部分 EMT 的诱导。完全 EMT 的特征是 E-钙粘蛋白下调和 ZEB2 上调。部分 EMT 的特征是 E-钙粘蛋白上调和波形蛋白和 ZEB2 下调。有趣的是，我们发现 Slug 通过下调 ABC 转运蛋白 ABCB1 和上调 ABC 转运蛋白 ABCG2 以及通过表达 CD133（一种干性标志物）诱导化疗耐药，CD133 的表达模式与具有完全或部分 EMT 表型的细胞相似。此外，我们发现 Slug 介导的部分 EMT 与外泌体分泌的翻译后修饰纤维连接蛋白 1（FN1）、胶原 II 型 alpha 1（COL2A1）和天然纤维蛋白原 gamma 链（FGG）增加有关。
根据我们的数据，我们得出结论，鉴定的外泌体蛋白可被视为 HCC 中化疗耐药和部分 EMT 的潜在非侵入性生物标志物。
