网状蛋白-4家族与肾癌的关系及其分子机理研究

Globally, about 270,000 cases of renal cancer (RC) are diagnosed yearly and 110,000 patients die from this disease. Adult renal cancers that arise in the renal parenchyma are mainly adenocarcinomas, also known as renal cell carcinomas (RCCs), accounting for more than 90% of adult kidney carcimomas. Despite advances in diagnosis, about 20-30% of the patients are diagnosed with metastatic disease. Another 20% of patients undergoing nephrectomy will have a relapse and develop metastatic RCC (mRCC) during follow-up. The prognosis for patients with mRCC is extremely poor, making the occurrence of mRCC a serious problem for oncologic health care around the world. The causes of RCC are poorly understood. Genetic factors and their interaction with enviromental exposures are believed to influence risk of developing RCC. Reticulon-4(Rtn-4) is encoded by a single gene on human chromosome 2 (2p16) with 14 exons spanning a 75 Kb stretch and 10 known splice variants. The three major splice variants are Rtn-4A, Rtn-4B and Rtn-4C. Rtn-4 is almost exclusively found in the peripheral, reticular endoplasmic reticulum (ER). Growing evidence suggests that Rtn-4 is multi-functional, regulating apoptosis, tumor development, and neuronal regeneration. Rtn-4C overexpression in HEK293 cell confers to apoptosis by inducing caspase-3 and p53 activation through the JNK-c-Jun-dependent pathway. Two coordinated responses are activated in response to Rtn-4B overexpression, severe ER stress induced by high Rtn-4B overexpression causes apoptosis through ER Ca2+ depltion, NF-κB activation, CHOP activation and ER-specfic apoptotic pathways. At the same time, Rtn-4B overexpression activated the protective response to adapt and increase the resistance to ER-stress-associated stimuli. The novel role of Rtn-4A in promoting neuronal survival by controlling Rac1/RhoA balance has also been identified. In our pilot study, the association between the TATC insertion/deletion polymorphism in the 3'-UTR region of Rtn-4 gene and RCC has been identified. We screened mutations of Rtn-4 gene in a small number of RCC patients and identified three novel mutations, which were E250K，E278V, and D288G. Expression of Rtn-4B mRNA in RCC tissue is significantly higher than that in controls. While Western Blot results shown that Rtn-4 protein expression in RCC tissue decreased. The expression of NgBR mRNA has also significantly decreased in RCC tissue. Our results suggested that Rtn-4 may play a very important role in the pathogenesis of RCC. The present program will study the association between Rtn-4 and RCC by mutation screening in RCC patients. The effect of Rtn-4 mutations will be characterized by Western Blot immunohistochemistry, qRT-PCR, and so on. The molecular mechanism will also be explored. The results may contribute to our knowledge on the pathogenesis of RCC and to the research on RCC treatment.

肾癌是常见的泌尿系统肿瘤，其发病机制研究是该领域研究的难点和重点。网状蛋白-4（Rtn-4）有Rtn-4A、B及C三个主要成员，经MAPK(JNK/SAPK、p38)、NF-κB信号通路，调控细胞凋亡。课题组前期研究发现Rtn-4基因3'-UTR区TATC插入/缺失多态性位点与肾癌相关，在小样本肾癌患者中发现Rtn-4基因E250K，E278V及D288G三处新突变位点，肾癌组织中Rtn-4B mRNA表达增高，蛋白表达降低，受体NgBR mRNA表达降低，从多角度提示Rtn-4与肾癌密切相关。本研究拟从DNA遗传变异、mRNA和蛋白表达、蛋白亚细胞定位等角度，全面深入研究Rtn-4家族与肾癌发生发展的关系及分子机制、其作为肾癌标记物的可行性，采用质粒转染、RNA干扰使靶基因高表达、失表达，研究其对肾癌细胞死亡/存活的影响及其信号通路，探索干预Rtn-4表达调控抑制肾癌发生发展的新途径。

肾癌是常见的泌尿系统肿瘤，其发病机制研究是该领域研究的难点和重点。基因突变与肿瘤发生发展密切相关。本项目研究发现RTN4基因3’-UTR的TATC及CAA插入/缺失多态性位点与肾癌发病密切相关；RTN4基因mRNA和蛋白表达在癌组织和癌旁组织间有显著性差异；采用DHPLC技术方法在肾癌患者组中筛查到RTN4基因E250K、E278V、D288G和M380T等四个新突变位点；构建了以pcDNA3.1(+)为载体的RTN4基因野生型（WT）、E250K、E278V、D288G及M380T突变型质粒，转染ACHN细胞后筛选出相应的基因过表达稳转株；采用CRISPR/Cas9技术构建了RTN4基因敲除/敲低ACHN细胞模型；E250K及E278V突变促进细胞增殖；WT组中AKT mRNA表达下调而蛋白表达上调、NF-κB和p38均上调；而E250K、E278V等突变显著促进p38和NF-κB，抑制AKT。我们的研究结果表明，肾癌患者中新发现的E250K、E278V、D288G和M380T为功能性突变，通过p38 MAPK和NF-κB途径，促进细胞增殖，从而参与肾癌发生发展。