von Hippel-Lindau tumor suppressor gene-dependent mRNA stabilization of the survival factor parathyroid hormone-related protein in human renal cell carcinoma by the RNA-binding protein HuR
von Hippel-Lindau tumor suppressor gene-dependent mRNA stabilization of the survival factor parathyroid hormone-related protein in human renal cell carcinoma by the RNA-binding protein HuR
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DOI：
10.1093/carcin/bgn275
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发表时间：
2009-03-01
期刊：
影响因子：
4.7
通讯作者：
Massfelder, Thierry
中科院分区：
文献类型：
--
作者：
Danilin, Sabrina;Sourbier, Carole;Massfelder, Thierry
我们已经证明，甲状旁腺激素相关蛋白(PTHrP)是人肾细胞癌(RCC)的生存因子，其表达受von Hippel-Lindau(VHL)抑癌基因在信使RNA(MRNA)稳定水平上的负调控，与肿瘤生长因子(TGFs)一样。我们的目标是分析人肾细胞癌中PTHrP mRNA的选择性剪接，并从这些结果中确定VHL/缺氧诱导因子(HIF)系统调节的与PTHrP mRNA稳定性有关的mRNA结合蛋白。我们使用：(I)一组表达或不表达VHL的人RCC细胞；(Ii)VHL缺陷的786-0细胞，转染活性或非活性VHL；(Iii)人RCC样本和相应的正常组织。通过实时定量逆转录-聚合酶链式反应分析，发现141个PTHrP亚型在所有细胞和肿瘤中占主导地位(80%)。在VHL转染的细胞中，所有异构体的表达均降低了50%。8种蛋白质的相对分子质量在20~75 kDa之间，它们与跨越141个PTHrP基因富含AU的3‘非翻译区的生物素化转录本结合，其丰度依赖于VHL的表达。该蛋白的表观分子量为30 kDa，经免疫印迹鉴定为Hur，是一种RNA结合蛋白，具有稳定多种mRNA的功能，编码多种与恶性转化有关的蛋白质，包括血管内皮生长因子和转化生长因子-β。PTHrP的表达研究证实，Hur参与了PTHrP在本病中的上调。受VHL/HIF系统调控的常见的mRNA结合蛋白可能构成针对仍然难以治疗的人肾细胞癌的新的治疗机会。
We have shown that parathyroid hormone-related protein (PTHrP) is a survival factor for human renal cell carcinoma (RCC) and that its expression is negatively regulated by the von Hippel-Lindau (VHL) tumor suppressor gene at the level of messenger RNA (mRNA) stability, as observed for tumor growth factors (TGFs). Our goals were to analyze the alternative splicing of PTHrP mRNA in human RCC and from these results to identify VHL/hypoxia-induced factor (HIF) system-regulated mRNA-binding proteins involved in PTHrP mRNA stability. We used: (i) a panel of human RCC cells expressing or not VHL; (ii) VHL-deficient 786-0 cells transfected with active or inactive VHL and (iii) human RCC samples and corresponding normal tissues. By quantitative real-time reverse transcription-polymerase chain reaction analysis, the 141 PTHrP mRNA isoform was found to be predominant in all cells and tumors (80%). In cells transfected with VHL, the expressions of all isoforms were decreased by 50%. Eight proteins with molecular weights ranging from 20 to 75 kDa were found to bind to biotinylated transcripts spanning the 141 PTHrP mRNA AU-rich 3'-untranslated region whose abundancy was dependent on VHL expression. The protein having an apparent molecular weight of 30 kDa was identified by western blot as HuR, a RNA-binding protein with stabilizing functions on various mRNA coding for proteins important in malignant transformation including vascular endothelial growth factor and TGF-beta. PTHrP expression studies confirmed the involvement of HuR in PTHrP upregulation in this disease. Common mRNA-binding proteins regulated by the VHL/HIF system may constitute new therapeutic opportunities against human RCC that remains refractory to therapies.