Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
Core Research Facility, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
Int J Mol Sci. 2021 Nov 23;22(23):12647. doi: 10.3390/ijms222312647.
The aim of this study was to analyze the biological role of different transforming growth factor-β (TGFβ) receptor splice variants in ovarian carcinoma (OC). Specific receptor variant knockouts (KO) were prepared using the CRISPR/Cas9 genome editing system in two OC cell lines, TβRI variant 1 (TβRIv1) KO in ES-2 cells and TβRII variant 1 (TβRIIv1) KO in OVCAR-8 cells. Control and KO cells were compared by proteomic analysis, functional tests, analysis of epithelial-mesenchymal transition (EMT) drivers, and Western blot of signaling proteins. Proteomic analysis revealed significant changes in protein pathways in the KO cells. TβRIv1 KO resulted in a significant reduction in both cellular motility and invasion, while TβRIIv1 KO significantly reduced cellular motility and increased Reactive Oxygen Species (ROS) production. Both receptor variant KOs reduced MET protein levels. Of the EMT drivers, a significant decrease in TWIST protein expression, and increase in SNAIL protein and mRNA levels were observed in the TβRIIv1 KO compared to control. A significant decrease in JNK1 and JNK2 activation was found in the TβRIv1 KO compared to control cells. These findings provide new insight regarding the biological role of the TGFβ receptor variants in the biology and potentially the progression of OC.
本研究旨在分析不同转化生长因子-β（TGFβ）受体剪接变体在卵巢癌（OC）中的生物学作用。使用 CRISPR/Cas9 基因组编辑系统在两种 OC 细胞系中制备了特定的受体变体敲除（KO），即 ES-2 细胞中的 TβRI 变体 1（TβRIv1）KO 和 OVCAR-8 细胞中的 TβRII 变体 1（TβRIIv1）KO。通过蛋白质组学分析、功能测试、上皮-间充质转化（EMT）驱动因子分析以及信号蛋白的 Western blot 比较对照和 KO 细胞。蛋白质组学分析显示 KO 细胞中的蛋白途径发生了显著变化。TβRIv1 KO 导致细胞迁移和侵袭能力显著降低，而 TβRIIv1 KO 则显著降低细胞迁移能力并增加活性氧物种（ROS）的产生。两种受体变体 KO 均降低了 MET 蛋白水平。在 EMT 驱动因子中，与对照相比，TβRIIv1 KO 中 TWIST 蛋白表达显著降低，SNAIL 蛋白和 mRNA 水平升高。与对照细胞相比，TβRIv1 KO 中 JNK1 和 JNK2 的激活显著降低。这些发现为 TGFβ 受体变体在 OC 生物学和潜在进展中的生物学作用提供了新的见解。
