长链非编码RNALINC01589介导LASP1调控PI3K/AKT信号通路抑制胆囊癌侵袭转移的机制研究

Gallbladder cancer (GBC) has a very poor prognosis. The incidence of GBC in China ranks the world. Recently, mounting evidences have shown that long non-coding RNAs (lncRNAs) are hot topic in cancer research. Yet, few studies about lncRNAs and GBC were reported. In our previous study, an unreported lncRNA "LINC01589" was found significantly down-regulated in gallbladder cancer tissue, and the expression level of it was associated with prognosis and the number of copies of the genome. After defining its basic characteristics and genomic localization, it was found that the knockout of LINC01589 could promote the invasion and metastasis of gallbladder cancer cells in vitro and in vivo. The molecular mechanism study showed that the knockout of LINC01589 significantly enhanced the phosphorylation level of the direct binding protein LASP1 and promoted it transportation to the nucleus. The antitumor effect of LINC01589 is correlated with PI3K/AKT signaling pathway. This project will deeply explore the effect of LINC01589 on invasion and metastasis of gallbladder cancer cell, the molecular basis of LINC01589 combing with LASP1 and the regulatory mechanism of LINC01589/LASP1 axis targeting genes and downstream signal pathway PI3K/AKT from the view of cell and molecular level in vivo and in vitro. The aim of this study is to clarify the function and molecular mechanism of LINC01589 in GBC invasion and metastasis, which could provide new targets for therapy.

胆囊癌预后极差，在我国发病率居世界前列。长链非编码RNA（lncRNA）是肿瘤研究的热点，但涉及胆囊癌的研究少且局限。我们前期研究发现一条尚未报道的lncRNA“LINC01589”在胆囊癌组织中表达显著下调，表达水平和预后以及基因组拷贝数相关。明确其基本特征和基因组定位后，体内外实验发现敲除LINC01589能促进胆囊癌细胞侵袭转移，反之则抑制。分子机制研究表明：敲除LINC01589能显著提高其直接结合蛋白LASP1的磷酸化水平、促进后者入核；其抑癌作用与PI3K/AKT信号通路相关。本项目拟通过体内外实验，在细胞与分子水平深入研究LINC01589对胆囊癌细胞侵袭转移能力的影响、LINC01589与LASP1结合的分子基础、LINC01589/LASP1轴对靶基因及下游信号通路PI3K/AKT的调控机制，旨在阐明LINC01589在胆囊癌侵袭转移中的功能及分子机制，为治疗提供新靶点。

胆囊癌是消化系统中较为常见的恶性肿瘤，其具有起病隐匿、转移能力强的特点，常规的辅助治疗手段在胆囊癌中均效果不佳。长链非编码RNA是肿瘤研究的热点，其在多种肿瘤的发生发展中发挥了重要的作用。在胆囊癌中针对lncRNA的研究较少，且缺乏原创性。我们通过lncRNA芯片筛选，我们得到在胆囊癌组织中差异表达显著的4条lncRNA，并通过RT-qPCR初步验证后选择差异表达最显著的LINC01589。在80对胆囊癌及癌旁组织标本中，LINC01589在肿瘤组织中的表达水平显著降低，且低表达LINC01589的胆囊癌患者预后不佳。通过3’RACE实验和5’RACE实验以及Northern Blot实验，证实LINC01589在胆囊癌中主要存在形式为一段593nt的序列。核质分离的RT-qPCR实验及RNA-FISH实验证实，LINC01589在胆囊癌细胞中主要存在位置为细胞质。Transwell迁移实验和侵袭实验证实，敲除LINC01589后胆囊癌细胞迁移能力和侵袭的能力增强，裸鼠体内转移模型同样证实，敲除LINC01589增强胆囊癌转移能力。RNA pulldown实验及对差异条带的质谱分析初步提示，LINC01589可能与LASP1蛋白结合，而进一步的Western Blot实验和RIP实验则证实LINC01589可以与LASP1特异性结合。核质分离的Western Blot实验证实，敲除LINC01589后LASP1蛋白在细胞核内的分布增加。对PI3K-AKT信号通路关键基因及蛋白的RT-qPCR实验及Western Blot实验提示，敲除LINC01589后PI3K-AKT通路被显著激活。通过GENEmania数据库，TCGA数据库及STRING数据库分析，我们猜测LASP1入核后可能通过介导斑联蛋白激活PI3K-AKT信号通路，进而促进胆囊癌侵袭转移。LINC01589在胆囊癌组织中表达水平显著降低，低表达LINC01589提示胆囊癌预后不佳。敲除LINC01589可以促进胆囊癌的侵袭转移过程。LINC01589可与LASP1蛋白特异性结合。在胆囊癌中LINC01589表达水平降低导致其结合蛋白LASP1由胞质入核增多，并可能通过介导斑联蛋白激活PI3K-AKT信号通路，进而促进胆囊癌侵袭转移。