Department of Breast Surgery, Affiliated Fuzhou First Hospital of Fujian Medical University, Fuzhou, 350009, China.
Department of Gynaecology, Affiliated Fuzhou First Hospital of Fujian Medical University, Fuzhou, 350009, China.
Cell Mol Biol (Noisy-le-grand). 2022 May 31;68(5):192-201. doi: 10.14715/cmb/2022.68.5.26.
It was to investigate the regulation of motigen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathways mediated by epithelial cell adhesion molecules (EpCAM) N-glycosylation on epithelial-mesenchymal transition (EMT) in breast cancer cells. Breast cancer cells MCF-7 and MDA-MB-231 were taken in the control group; the blank vector plasmid transfection (blank plasmid group) and EpCAM N-glycosylation mutant plasmid transfection (Mutant-EpCAM group) were analyzed. The EpCAM in breast cancer cells was localized by immunofluorescence technique, the proliferation activity of cells in each group was detected by methyl thiazolyl tetrazolium (MTT) assay, and the clonality of cells was detected by plate cloning. and apoptosis-related proteins (Caspase-3, Bcl-2, and Bax), EMT-related molecular markers (E-cadherin, N-cadherin, and Vimentin), as well as MAPK and PI3K/Akt pathway-related proteins (p38, PI3K, and Akt) in cells were detected by western blotting (WB). EpCAM N-glycosylation mutations did not alter the expression localization of EpCAM in breast cancer cells. Compared with the control group and the blank plasmid group, the cell proliferation activity and the number of colonies formed were decreased in the Mutant-EpCAM group (P<0.05). The protein expressions of Caspase-3, Bax, and E-cadherin were up-regulated significantly, and the expressions of Bcl-2, N-cadherin, Vimentin, p-p38, p-PI3K, and p-Akt were greatly down-regulated (P<0.05). EpCAM N-glycosylation could regulate the EMT in breast cancer cells through MAPK and PI3K/Akt pathways.
目的在于研究上皮细胞黏附分子（EpCAM）N-糖基化对乳腺癌细胞上皮-间质转化（EMT）中促分裂原激活的蛋白激酶（MAPK）和磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶 B（Akt）通路的调节作用。选取乳腺癌细胞 MCF-7 和 MDA-MB-231 作为对照组；转染空白载体质粒（空白质粒组）和 EpCAM N-糖基化突变质粒（Mutant-EpCAM 组）进行分析。采用免疫荧光技术定位乳腺癌细胞中的 EpCAM，采用噻唑蓝（MTT）比色法检测各组细胞的增殖活性，采用平板克隆实验检测细胞的克隆性。采用 Western blot（WB）检测细胞中凋亡相关蛋白（Caspase-3、Bcl-2 和 Bax）、EMT 相关分子标志物（E-cadherin、N-cadherin 和 Vimentin）以及 MAPK 和 PI3K/Akt 通路相关蛋白（p38、PI3K 和 Akt）。EpCAM N-糖基化突变并未改变乳腺癌细胞中 EpCAM 的表达定位。与对照组和空白质粒组相比，Mutant-EpCAM 组细胞增殖活性和集落形成数均降低（P<0.05）。Caspase-3、Bax 和 E-cadherin 的蛋白表达显著上调，Bcl-2、N-cadherin、Vimentin、p-p38、p-PI3K 和 p-Akt 的表达明显下调（P<0.05）。EpCAM N-糖基化可通过 MAPK 和 PI3K/Akt 通路调节乳腺癌细胞 EMT。
