Department of Urology, Chinese People's Liberation Army General Hospital, Beijing, P.R. China.
Urol Oncol. 2013 Aug;31(6):938-48. doi: 10.1016/j.urolonc.2011.07.006. Epub 2011 Oct 10.
Although NOTCH1 plays a wide-ranging role in controlling cell fate, differentiation, and development, its pathologic roles in clear cell renal cell carcinoma (CCRCC) are still unclear. In the present study, the expression pattern of NOTCH1 was examined in CCRCC tissues, and the interaction of NOTCH1 with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/phosphatidylinositol 3-kinase (PI3K)/AKT pathway was investigated in vitro.
Thirty-six paired CCRCC and adjacent non-neoplastic renal samples were analyzed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The alteration of NOTCH1, hairy and enhancer of split 1 (HES1), PTEN, AKT (phosphorylated at Ser473) in CCRCC cell line (786-O), and the human normal kidney tubule epithelial cell line (HKC) were analyzed by Western blotting and qRT-PCR, before and after transfection with siRNA against NOTCH1 or the plasmid containing the ORF clone of NOTCH1. The effects of NOTCH1 signaling pathway on cells proliferation, apoptosis, invasion, and migration were detected by MTS assay, flow cytometry analyses, and transwell chamber assay, respectively.
The NOTCH1 expression levels were significantly increased in CCRCC tissues compared with the adjacent non-neoplastic renal samples, while it had no significant association with the pathologic parameters. NOTCH1 signaling cascade was constitutively active in human CCRCC cell lines. Blocking NOTCH1 signaling resulted in the attenuation of proliferation, invasion, and migration, as well as PTEN up-regulation with decreased AKT phosphorylation. NOTCH1 overexpression had an opposite effect to NOTCH1 knockdown.
Our findings indicated that NOTCH1 receptor expression was up-regulated in CCRCC, and that NOTCH1 could regulate PTEN expression and the activity of the PI3K/AKT pathway via HES1 in 786-O and HKC cell lines. These might provide a basis for the designing new therapeutic strategies for CCRCC.
尽管 NOTCH1 在控制细胞命运、分化和发育方面发挥着广泛的作用，但它在透明细胞肾细胞癌（ccRCC）中的病理作用仍不清楚。在本研究中，我们检测了 NOTCH1 在 ccRCC 组织中的表达模式，并在体外研究了 NOTCH1 与磷酸酶和张力蛋白同源物缺失的第 10 号染色体（PTEN）/磷脂酰肌醇 3-激酶（PI3K）/AKT 通路的相互作用。
通过 Western blot 和实时定量聚合酶链反应（qRT-PCR）分析 36 对 ccRCC 和相邻非肿瘤性肾组织样本。通过 Western blot 和 qRT-PCR 分析 ccRCC 细胞系（786-O）和人正常肾小管上皮细胞系（HKC）中 NOTCH1、头发和增强子分裂 1（HES1）、PTEN、AKT（丝氨酸 473 磷酸化）的改变，然后用 NOTCH1 或包含 NOTCH1 ORF 克隆的质粒转染。通过 MTS 测定、流式细胞术分析和 Transwell 室测定分别检测 NOTCH1 信号通路对细胞增殖、凋亡、侵袭和迁移的影响。
与相邻的非肿瘤性肾组织样本相比，ccRCC 组织中 NOTCH1 的表达水平显著升高，但其与病理参数无显著相关性。NOTCH1 信号级联在人 ccRCC 细胞系中持续激活。阻断 NOTCH1 信号导致增殖、侵袭和迁移减弱，同时 PTEN 上调，AKT 磷酸化减少。NOTCH1 过表达的效果与 NOTCH1 敲低相反。
我们的研究结果表明，NOTCH1 受体在 ccRCC 中表达上调，NOTCH1 可以通过 HES1 在 786-O 和 HKC 细胞系中调节 PTEN 表达和 PI3K/AKT 通路的活性。这可能为设计 ccRCC 的新治疗策略提供依据。
