Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Department of Urology, Jiangsu Province Hospital of TCM, Affiliated Hospital of Nanjing University of TCM, Nanjing, 210029, China.
J Exp Clin Cancer Res. 2018 Feb 27;37(1):40. doi: 10.1186/s13046-018-0706-6.
Wilms' tumor 1-associating protein (WTAP) plays an important role in physiological processes and the development of tumor such as cell cycle regulation. The regulation of cell cycle is mainly dependent on cyclins and cyclin-dependent protein kinases (CDKs). Recent studies have shown that CDKs are closely related to the tumor diagnosis, progression and response to treatment. However, their specific biological roles and related mechanism in renal cell carcinoma (RCC) remain unknown.
Quantitative real-time PCR, western blotting and immunohistochemistry were used to detect the expression of WTAP and CDK2. The survival analysis was adopted to explore the association between WTAP expression and the prognosis of RCC. Cells were stably transfected with lentivirus approach and cell proliferation and cell cycle, as well as tumorigenesis in nude mice were performed to assess the effect of WTAP in RCC. RNA immunoprecipitation, Luciferase reporter assay and siRNA were employed to identify the direct binding sites of WTAP with CDK2 transcript. Colony formation assay was conducted to confirm the function of CDK2 in WTAP-induced growth promoting.
In RCC cell lines and tissues, WTAP was significantly over-expressed. Compared with patients with low expression of WTAP, patients with high expression of WTAP had lower overall survival rate. Additionally, cell function test indicated that cell proliferation abilities in WTAP over-expressed group were enhanced, while WTAP knockdown showed the opposite results. Subcutaneous xenograft tumor model displayed that knockdown of WTAP could impede tumorigenesis in vivo. Mechanism study exhibited that CDK2 expression was positively associated with the expression of WTAP. Moreover, WTAP stabilized CDK2 transcript to enhance CDK2 expression via binding to 3'-UTR of CDK2 transcript. Additionally, specific inhibitors of CDK2 activity and small interfering RNA (siRNA) of CDK2 expression inhibited WTAP-mediated promotion of proliferation.
These findings suggest that WTAP may have an oncogenic role in RCC through physically binding to CDK2 transcript and enhancing its transcript stability which might provide new insights into RCC therapy.
Wilms' 肿瘤 1 相关蛋白（WTAP）在细胞周期调控等生理过程和肿瘤发生发展中发挥重要作用。细胞周期的调控主要依赖于细胞周期蛋白和细胞周期蛋白依赖性激酶（CDK）。最近的研究表明，CDK 与肿瘤的诊断、进展和治疗反应密切相关。然而，它们在肾细胞癌（RCC）中的具体生物学作用及相关机制尚不清楚。
采用实时定量 PCR、western blot 及免疫组化方法检测 WTAP 和 CDK2 的表达。采用生存分析探讨 WTAP 表达与 RCC 预后的关系。采用慢病毒转染技术稳定转染细胞，观察 WTAP 对 RCC 细胞增殖、细胞周期及裸鼠成瘤的影响。采用 RNA 免疫沉淀、荧光素酶报告基因检测及 siRNA 技术鉴定 WTAP 与 CDK2 转录本的直接结合位点。采用集落形成实验证实 CDK2 在 WTAP 诱导的促生长中的作用。
在 RCC 细胞系和组织中，WTAP 表达明显上调。与 WTAP 低表达的患者相比，WTAP 高表达的患者总生存率较低。此外，细胞功能试验表明，WTAP 过表达组细胞增殖能力增强，而 WTAP 敲低组则表现出相反的结果。皮下移植瘤模型显示，WTAP 敲低可抑制体内肿瘤的发生。机制研究表明，CDK2 的表达与 WTAP 的表达呈正相关。此外，WTAP 通过与 CDK2 转录本的 3'-UTR 结合稳定 CDK2 转录本，从而增强 CDK2 的表达。此外，CDK2 活性的特异性抑制剂和 CDK2 表达的小干扰 RNA（siRNA）抑制了 WTAP 介导的增殖促进作用。
这些发现表明，WTAP 通过与 CDK2 转录本物理结合并增强其转录稳定性，在 RCC 中可能发挥致癌作用，这可能为 RCC 的治疗提供新的思路。
