长链非编码RNA GAS5调控基因翻译的分子机制及其在膀胱癌发展中的作用

Bladder cancer is the most common malignant tumor of urinary system. Because of unknown pathogenesis, there is an urgent need for revealing the molecular mechanisms of the initiation and development of bladder cancer . Growth arrest-specific 5 (GAS5) is a long non-coding RNA with the function of regulation cell survival. Recently, the expression of GAS5 has been proved significantly decreased in tumors, however, the precise signaling pathway remains largely unknown. In present study, we found that GAS5 levels were highly related with the pathology grade and TNM stage of bladder cancer. We found that the GAS5 can interact with eukaryotic translation initiation factor eIF4E, participating in the control of gene translation. Moreover, the interaction between GAS5 and eIF4E is much weaker in bladder cancer cell line. The eIF4E was also found to be partly involved in selective translational control mediated by GCN2 signaling. Hence, it is important to understand the translational control mechanisms of GAS5 for the development of bladder cancer. In this project, we will expand the bladder cancer samples to determine the clinical significance of GAS5. We will also establish cell models to study the function of GAS5 in bladder carcinoma development. Then, we will screen the important interaction proteins and key effector mRNAs of GAS5, through specific RNA pull down and RNA binding protein immunoprecipitation techniques. This research will elucidate the specific translational control by GAS5 in the development of bladder cancer, and provide more potential targets and ample basis for clinical therapy of bladder cancer.

膀胱癌是最常见的泌尿系统恶性肿瘤。由于发病机制不甚清楚，我们迫切需要揭示膀胱肿瘤发生发展的分子机制。长链非编码RNA Growth arrest-specific 5（GAS5）能够调控细胞的生存和死亡，近年来研究发现它在多种肿瘤中表达降低，但机制研究不多。我们发现GAS5的表达与临床膀胱癌的恶性程度相关。细胞质中的GAS5与真核翻译起始因子eIF4E结合，且该相互作用在膀胱癌细胞中明显减弱。eIF4E能参与GCN2压力感受信号介导的选择性翻译抑制。由此我们提出存在GAS5选择性调控基因翻译的分子机制，直接影响膀胱癌的发展。本课题将扩大临床膀胱癌样本的检测，并建立细胞模型研究GAS5在膀胱癌中的功能；通过免疫共沉淀技术，确定GAS5相互作用的蛋白并筛选其调控的关键效应mRNA。本研究将阐述GAS5如何通过翻译调控影响膀胱癌发展，为临床治疗研究提供更多靶点和数据基础。

泌尿系统肿瘤的复发和进展是临床难题。膀胱癌组织中长链非编码RNA Growth arrest-specific 5（GAS5）表达明显下调。较低的GAS5表达水平与晚期病理阶段和较差的总生存率有关。LncRNA GAS5在膀胱癌细胞T24、5637和SW780中的表达也低于正常的人尿路上皮细胞系(Sv-Huc-1)。吉西他滨（gemcitabine）是膀胱癌常用的化疗药物，GAS5水平与癌细胞对吉西他滨的敏感性密切相关。吉西他滨可剂量依赖性地增加膀胱癌细胞的GAS5水平。值得注意的是，预处理吉西他滨对第二剂吉西他滨诱导的GAS5表达有一定的抑制作用。过表达GAS5的细胞显示吉西他滨诱导的细胞死亡增多。因此，通过小干扰RNA敲低膀胱癌细胞中的GAS5，使其对吉西他滨诱导的细胞毒性具有耐受性。脱氧胞苷激酶（deoxycytidine kinase，DCK）是负责吉西他滨转化为活性抗代谢物的限速酶。通过敲除膀胱癌细胞中的GAS5，证实GAS5调控DCK的表达。此外，分析GAS5相互作用的RNA结合蛋白，我们还发现GAS5的促凋亡功能与促进HuR出核和DCK表达的生物学效应有关。本研究表明，长链非编码RNA GAS5可调节HuR介导的DCK表达，而GAS5低表达可耐受吉西他滨。我们证明了长链非编码RNA GAS5调控吉西他滨敏感性的能力，GAS5的表达可能是一种新的辅助诊断和破坏化疗耐药性的靶点。我们在研究过程中发现翻译调控激酶（General control nonderepressible kinase 2，GCN2)可以调控肾癌靶向药物的敏感性。相当比例的肾细胞癌患者天生对索拉非尼耐受，其余患者大多会出现耐药。索拉非尼增加GCN2及其下游eIF2α的磷酸化激活。沉默GCN2表达也可促进索拉非尼诱导的细胞凋亡。我们发现抗凋亡基因环氧化酶2 (cyclooxygenase 2，cox2)在索拉非尼耐药细胞中表达较高。为了克服索拉非尼的耐药性，我们使用临床用cox2抑制剂塞来昔布。体外和体内实验证明，低剂量的塞来昔布和索拉非尼联合治疗ACHN细胞比单独使用索拉非尼的效率更高。我们的研究结果表明，cox2抑制剂塞来昔布联合治疗可能是一种新的耐药肾细胞癌的治疗方法。研究结果为泌尿系统肿瘤的治疗提供了新的靶点，为逆转肿瘤耐药提供了新策略。