肾癌细胞中VHL失活对microRNA加工的影响

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene underlies the majority of renal cell cancers (RCC), and its loss of function results in constitutive stabilization of the transcription factor HIF (hypoxia-inducible factor). HIF (HIF-1a and -2a) activates the transcription of many genes, producing cell proliferation, angiogenesis, metastasis and other alterations, which altogether causes the development of RCC. HIF also induces the expression of different microRNAs that influence tumorigenesis. Besides inducing specific sets of microRNAs, tumors in general are known to bear alterations in microRNA processing, which allows the down-regulation of key microRNAs with tumor suppression function. In this proposal we aim to investigate the effects of VHL inactivation/HIF activation on the microRNA processing of RCC cells. Our preliminary data show that VHL inactivation in RCC cells reduces the expression of the microRNA processing proteins TRBP and Dicer. Our first objective will be to study how down-regulation of TRBP/Dicer influences the efficiency of microRNA processing in VHL-defective RCC cell lines engineered to re-express wild-type or mutated forms of VHL. In addition, we will test whether down-regulation of TRBP and Dicer influences RCC cell proliferation, metastasis and angiogenesis. Moreover, we will perform mechanistic studies to understand how VHL inactivation down-regulates TRBP/Dicer, with the specific hypothesis that the effects are mediated by HIF. Establishing a connection between VHL inactivation/HIF activation and the microRNA processing pathway will provide further understanding of how RCC tumors appear and progress, and considering that HIF is frequently activated in all types of cancer this may have as well widespread implications. Potentially, our work could also shed light into novel drug targets for RCC therapy.

VHL 基因失活是诱发肾癌最常见的因素之一，其失活可使HIF1/2的蛋白稳定性增强，使得肿瘤获得生长加快，周边血管生成，侵袭性增强等特征，加速肿瘤的发生和发展。microRNA对于靶基因的调控紊乱是肿瘤也是肾癌的一个重要特征，这种调控紊乱也可加速肿瘤的发生和发展。但是目前VHL失活对microRNA加工的影响尚未见报道。我们前期工作发现在VHL失活的肾癌细胞中，microRNA相关的加工蛋白（TRBP和Dicer）的表达显著下调。我们拟进一步通过microRNA实时定量PCR技术检测VHL失活对microRNA加工效率的影响。另外，我们外源过表达或者敲低TRBP和Dicer，研究它们对肾癌增殖、转移以及血管生成的影响。将肾癌中最常见的失活基因VHL和microRNA加工这两个信号通路联系起来，解释VHL失活对于肾癌发生发展的作用，甚至为肿瘤的治疗提供潜在的靶点。

肾癌是一种起源于肾脏的癌症，它主要由肾细胞癌和泌尿道上皮细胞癌组成，全球每年约有 208 500 例肾细胞癌被确诊,占所有癌症比例的 3%,其 死亡率高达 40%,是成年人泌尿系统最常见的恶性肿瘤。相对于其它癌症,肾细胞癌对化疗和放疗常不敏感,因而对肾细胞癌的治疗通常只能采用外科手术的方法。但外科手术本身存在很大的局限 性,术后复发的可能性高达 20%-40%,故而目前对肾细胞癌的治疗尚无有效的方法。寻找新的诊断标记和治疗靶点对于肾细胞癌的诊断及治疗具有重大意义。值得一提的是，VHL突变是肾癌中最常见的突变类型。.在本项目的执行过程中，我们意外地发现，大剂量的维生素c可以选择性地杀死VHL突变的肿瘤细胞，而这种敏感性来自细胞内VHL突变所引起的低氧诱导因子（HIF）的激活。更进一步的研究表明，在HIF激活的细胞中，HIF可以通过转录调控，使得细胞明显高表达GLUT1受体，该受体又可以显著地促进氧化形式的维生素c向细胞内的运输。最终导致细胞内产生大量的DNA损伤，消耗大量的ATP,使肿瘤细胞迅速走向死亡。该发现揭示了肿瘤细胞为何对高剂量的维生素c更为敏感，为维生素c治疗肿瘤提供了理论基础，相关文章发表在国际杂志 The Journal of Biological Chemistry上。.另外，我们还发现了一个受到低氧诱导因子调控的长链非编码RNA, lincRNA-p21能够显著地提高体细胞重编程的效率。在肿瘤细胞中，lincRNA-p21可以抑制VHL对HIF的降解，从而促进肿瘤的生长。然而，在我们的重编程系统中，我们发现lincRNA-p21作为一个新的路障抑制体细胞重编程。在机理层面, 我们首次发现 HNRNPK 可以招募表观遗传因子 SETDB1 去调控多能性基因的 H3K9me3 水平进而抑制基因转录,也可以通过DNMT1抑制多能性基因的甲基化水平，从而抑制重编程的效率。我们的研究成果不仅丰富了对 lncRNA 功能的认识、加深了对重编程机制的理解,更为重要的是首次揭示 p53 通过 lincRNA-p21 去调控多能性基因的染色质状态,和异染色质的形成与维持建立了 联系。