Han Qipeng, Han Fangzhu, Fan Yisheng, Lian Bowen, Xiao Jinyang, Sun Wei, Han Dongbing, Kou Hongbo, Li Chunyan, Wu Bin
Department of Urinary Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China.
Department of Urinary Surgery, General Hospital of Northern Theater Command, Shenyang, Liaoning 110001, P.R. China.
Oncol Lett. 2020 Dec;20(6):379. doi: 10.3892/ol.2020.12242. Epub 2020 Oct 22.
Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. Although deregulation of the Notch signaling pathway is common in RCC and is involved in the tumorigenic process, the exact role of Notch3 and its underlying molecular mechanism in RCC, particularly in hypoxia, remain unknown. In the present study, RO4929097, a Notch3 inhibitor, was used to alter NICD3 expression. A Cell Counting Kit-8 assay, EdU incorporation assay, colony formation assay, flow cytometry and western blot analysis were used to investigate the effects of altered NICD3 expression on cell proliferation, cell cycle progression and HIF-2α protein expression. The results of western blot analysis showed that RO4929097 dose-dependently decreased the expression of Notch3 intracellular domain (NICD3) in 786-O and ACHN cells, which originate from clear cell RCC (ccRCC). The results of the Cell Counting Kit-8, EdU incorporation and colony formation assays demonstrated that downregulation of NICD3 significantly suppressed cell proliferation in both normoxia and hypoxia. In addition, flow cytometry and western blot analysis demonstrated that hypoxia (2% O) promoted cell cycle progression in ccRCC cells with the increased expression of G-S transition-associated proteins, namely cyclin-dependent kinase (CDK)4 and cyclin D1, while downregulation of NICD3 exerted negative effects on cell cycle progression, and the expression levels of CDK4 and cyclin D1. Furthermore, western blot analysis revealed that 2% O-induced upregulated hypoxia-inducible factor-2α (HIF-2α) expression decreased following downregulation of NICD3 in 786-O and ACHN cells. Following transfection of the vector containing the NICD3 coding sequence, HIF-2α, CDK4, cyclin D1 and proliferating cell nuclear antigen expression, that were inhibited by RO4929097 in hypoxia, were rescued. Collectively, the results of the present study suggest that Notch3 is closely associated with the cell proliferation of ccRCC cells by regulating the cell cycle and HIF-2α.
肾细胞癌（RCC）是泌尿系统最常见的恶性肿瘤之一。尽管Notch信号通路失调在RCC中很常见且参与肿瘤发生过程，但Notch3在RCC中的具体作用及其潜在分子机制，尤其是在缺氧情况下，仍不清楚。在本研究中，使用Notch3抑制剂RO4929097来改变NICD3的表达。采用细胞计数试剂盒-8法、EdU掺入法、集落形成法、流式细胞术和蛋白质印迹分析来研究NICD3表达改变对细胞增殖、细胞周期进程和HIF-2α蛋白表达的影响。蛋白质印迹分析结果显示，RO4929097剂量依赖性地降低了源自透明细胞肾细胞癌（ccRCC）的786-O和ACHN细胞中Notch3细胞内结构域（NICD3）的表达。细胞计数试剂盒-8法、EdU掺入法和集落形成法的结果表明，NICD3的下调在常氧和缺氧条件下均显著抑制细胞增殖。此外，流式细胞术和蛋白质印迹分析表明，缺氧（2% O）通过增加G-S转换相关蛋白即细胞周期蛋白依赖性激酶（CDK）4和细胞周期蛋白D1的表达促进ccRCC细胞的细胞周期进程，而NICD3的下调对细胞周期进程以及CDK4和细胞周期蛋白D1的表达水平产生负面影响。此外，蛋白质印迹分析显示，在786-O和ACHN细胞中，NICD3下调后，2% O诱导的缺氧诱导因子-2α（HIF-2α）表达上调降低。转染包含NICD3编码序列的载体后，缺氧条件下被RO4929097抑制的HIF-2α、CDK4、细胞周期蛋白D1和增殖细胞核抗原的表达得到恢复。总的来说，本研究结果表明Notch3通过调节细胞周期和HIF-2α与ccRCC细胞的细胞增殖密切相关。
