TAp63/miR-200/Fascin信号通路调控膀胱癌细胞侵袭转移的机制研究

MicroRNAs (miRNAs) play important roles in tumor invasion and metastasis. Identification of tumor metastasis-related miRNAs and signaling pathway has become an important focus of cancer research.Our previous results confirmed the importance of Fascin in the development of bladder tumor and the potential of invasion and metastasis. Further investigation showed that activation of p63 downregulated the Fascin via induction of the miR-200 genes. candidate miRNAs(miR-200a/c) targeting the 3'-untranslated region of Fascin could inhibit Fascin expression and decrease the invasion of T24 cells . On the basis of these data, we hypothesized that TAp63 -mediated miR-200a/c expression may decrease the potential of invasion and migration of bladder cancer cells by directly down-regulating expression of Fascin. In this project, we aimed to a further investigate the direct targeted regulation of Fascin by TAp63/ miR-200a/c through a series of molecular biology methods such as dual-luciferase reporter assay, immunofluorescence localization, gene silencing, immunoblotting test and gene microarray. Furthermore, in vitro and in vivo experiments were conducted to illustrate the molecular mechanism underlying the inhibition of Fascin-conferred invasion and metastasis by TAp63/ miR-200a/c. The findings of this study may have important implications in demonstrating the mechanism of post-transcriptional regulation of Fascin and identification of new therapeutic targets for therapy for muscle-invasive bladder cancer.

鉴定以microRNA为核心的信号通路已成为肿瘤侵袭转移调控领域的核心问题。申请者在前期工作中阐明了成束蛋白（Fascin）在促进膀胱癌细胞恶性发展和侵袭迁移能力方面的重要作用，进而发现抑癌基因TAp63在转录活化miR-200的同时，也抑制了Fascin蛋白的表达。miR-200的"种子区域"序列与Fascin3'UTR 区序列完全匹配，而过表达miR-200a/c模拟物抑制Fascin表达并减少了T24细胞的侵袭迁移。由此我们提出假说：TAp63转录活化的miR-200a/c靶向下调Fascin表达，进而抑制了膀胱癌细胞侵袭转移。本研究拟进一步采用荧光素酶报告基因分析等分子生物学方法，结合功能实验，旨在获得TAp63/miR-200a/c 直接调控Fascin 相关侵袭转移信号通路的可靠证据。本项目将有助于阐明Fascin负性调控机制，为鉴定侵袭性膀胱癌的新治疗靶点提供充分的科学依据。

膀胱癌是泌尿系最常见的恶性肿瘤，其发生发展的分子机制尚不完全明确。由于对TAp63的研究出现困难，鉴于课题时间有限，我们调整了研究方向。我们经研究发现长链非编码RNA ZEB1-AS1在膀胱癌组织中较癌旁组织高表达，其在膀胱癌中可能作为一种癌基因而存在，而miR-200在多种肿瘤中是作为一种抑癌因子而存在，本研究旨在揭示膀胱癌中二者之间的调控关系。我们的结果显示ZEB1-AS1可以促进膀胱癌细胞的侵袭迁移、增殖，并可通过海绵吸附作用吸附miR-200，间接升高miR-200的靶基因FSCN1的表达进而发挥其促进细胞侵袭迁移的功能。本研究的结论是长链非编码RNA ZEB1-AS1可能通过miR-200/FSCN1信号通路对膀胱癌的发生发展起到促进的作用，为膀胱癌的诊治提供新的思路。