IPO11过表达上调THBS1促进膀胱尿路上皮癌侵袭进展的机制研究

The molecular mechanism of bladder urothelial carcinoma progression remains unclear. Importins, members of the karyopherin family, are a type of protein that transports cargo protein into the nucleus. Importins have been proven to be associated with the progression of various cancers. We have previously detected the DNA copy number amplification of IPO11 in basal tumors compared with their corresponding superficial tumors through whole exome sequencing in invasive bladder urothelial carcinoma. Further cytological experiments and immunohistochemical study have shown the overexpression of IPO11 mRNA and importin11 encoded by IPO11 in invasive bladder urothelial cancer. This overexpression is positively correlated with increased IPO11 copy number. IPO11 knockdown by RNAi technique inhibits the mobility and invasion of bladder cancer cell and downregulates THBS1 expression. Thus, in this work, we proposed a new molecular mechanism of progression in bladder urothelial cancer. In this mechanism, the overexpression of IPO11 induced by IPO11 DNA copy number amplification contributes to bladder urothelial carcinoma progression through the upregulation of transcription factor for THBS1. To disclose molecular mechanisms of bladder cancer invasion and progression induced by IPO11, we utilized technologies such as co-immunoprecipitation, ChIP, and RNAi to investigate the inter-reaction mechanism between IPO11 and its specific cargos and potential transcription factor binding sites on THBS1 gene through various methods, including cell biological function experiments and bioinformatics analysis. The aim of our study was to clarify the intra-nuclear mechanism of the IPO11 import signaling pathway and thus provide a new strategy for the prediction and target treatment of invasive bladder cancer.

非浸润性膀胱癌进展为浸润性癌的机制尚不明确，证据表明核转运受体中的输入蛋白（importin）与肿瘤进展关系密切。前期外显子组测序发现浸润性膀胱癌基底肿瘤相对其浅表肿瘤IPO11拷贝数扩增，细胞和免疫组化实验证实IPO11mRNA和其编码蛋白importin11在浸润性癌中高表达，并且IPO11拷贝数与蛋白表达水平有正相关趋势，沉默IPO11可以抑制膀胱癌细胞的迁移和侵袭能力，并且显著下调THBS1表达。由此提出假设：IPO11拷贝数扩增引起IPO11过表达，直接或间接使能够上调THBS1的转录因子在核内异常分布，诱导膀胱癌进展。本项目拟采用免疫共沉淀，质谱分析，ChIP，RNA干扰等技术，通过细胞生物学功能检测，生物信息学分析等方法阐释IPO11过表达上调THBS1的可能分子机制，探讨IPO11在其中发挥的作用，寻找可能的靶蛋白，为膀胱癌进展标志物及靶向药物的筛选提供理论和实践基础。

膀胱癌是我国发病率最高的泌尿系统肿瘤。低级别膀胱癌分化良好，高级别或肌层浸润癌分化差、容易进展和复发。为了探寻更好的膀胱癌诊断和治疗的有效方法，有必要进一步研究其发病机制。在本课题前期工作中，细胞和免疫组化实验证实IPO11mRNA和其编码蛋白importin11在浸润性癌中高表达，并且IPO11拷贝数与蛋白表达水平有正相关趋势，沉默IPO11可以抑制膀胱癌细胞的迁移和侵袭能力，并且显著下调THBS1表达。为了进一步研究IPO11发挥作用的机制，我们慢病毒构建IPO11稳定过表达的膀胱癌细胞株，建立Flag-IPO11的Co-IP检测体系，在IPO11过表达的T24细胞中经过蛋白CO-IP-质谱检测获得IPO11候选结合蛋白，经过筛选后通过CO-IP-WB实验进一步验证蛋白是否与IPO11相互作用，结果发现VIM（Vimentin）和CSE1L与IPO11相互作用，其他蛋白则为阴性结果。然后通过生物信息学分析预测THBS1相关的转录因子，将预测的 THBS1相关转录因子与CO-IP-质谱所得的IPO11相关蛋白进行交集分析，结果显示无交集蛋白。我们利用IPA互作网络分析，预测IPO11到THBS1的潜在转录因子路径，在路径中选择两条最可能途径，选取Jun与Tp53转录因子，并通过双荧光报告实验，结果显示，Jun与Tp53均不是THBS1的转录调控因子。同时，在功能基因数据库中，筛选到IPO11下游新功能基因IARS2，该基因参与蛋白翻译过程中tRNA的氨酰基化过程，在膀胱癌中，至今未见功能报道。通过RNAi手段在EJ细胞系中分别敲减IPO11及IARS2，qPCR和WB结果证明两者存在相关性。敲减IARS2后，对膀胱癌细胞的生长、增殖、侵袭迁移的能力有明显的抑制作用：敲减IARS2实验组细胞荧光计数显著低于对照组；5637细胞和EJ细胞系的CCK8细胞增殖实验中，敲减IARS2后，细胞增殖被显著抑制；在5637和EJ细胞系中，shIARS2组的克隆形成率显著低于对照组；在5637和EJ细胞系中，敲减IARS2显著促进细胞凋亡；敲减IARS2实验组EJ细胞的转移和侵袭能力受到显著抑制。而体内实验证明，敲减IARS2基因能够抑制膀胱癌细胞在小鼠体内的生长。因此IPO11和IARS2基因有望成为膀胱癌诊断、预后判断的新因子和治疗的新工具。