LncRNA-TC0101441抑制KiSS-1促进卵巢癌侵袭转移的作用及分子机制

The survival rate of ovarian cacer is poor. Such an unfavourable prognosis has been largely correlated with the tumour metastasis. The applicant had ever identified a new lncRNA (lncRNA-TC0101441) and found that TC0101441 contributed to E2-induced ovarian cancer cell invasion/migration. Recently, our preliminary experiments showed that TC0101441 correlated with poor prognosis of ovarian cancer, promoted cell invasion/migration and inhibited the expression of its target gene KiSS-1 in ovarian cancer cells. Based on these observations, in this project, we will use human clinical samples, in vitro studies and in vivo animal models to elucidate that TC0101441 can promote ovarian cancer cell invasion/migration partially through the suppression of its target gene KiSS-1 and that both TC0101441 and KiSS-1 may serve as independent predictors for overall survival. Moreover, we will demonstrate that TC0101441 can interact with Sp1 to form transcription complexes at specific Sp1-binding sites of the promoter to inhibit KiSS-1 transcription and explore the underlying molecular mechanism. These original researches may shed a new insight into the mechanism of ovarian cancer metastasis by providing a perspective of lncRNA and provide a rationale for the development of lncRNA-based targeted approaches for the treatment and prognosis estimation of ovarian cancer.

卵巢癌转移是影响预后的关键。申请人前期发现的新长链非编码RNA（lncRNA-TC0101441）能介导雌激素的促卵巢癌转移效应。新近实验进一步发现TC0101441与卵巢癌不良预后有关，促进卵巢癌的侵袭转移；且与临近靶基因KiSS-1表达呈负相关。在此基础上，本项目拟：在临床、细胞、动物水平，阐明TC0101441通过抑制KiSS-1促进卵巢癌侵袭转移的作用；分析TC0101441、KiSS-1成为预测卵巢癌预后、复发指标的可能性；通过截取不同长度启动子序列、构建野生及突变型载体、荧光素酶报告基因实验、RNA pulldown与质谱分析、RIP、EMSA及ChIP等技术，阐明TC0101441干扰Sp1对KiSS-1的转录调控，抑制KiSS-1表达的分子机制。这些原创性研究将从lncRNA角度为卵巢癌转移阐释新的调控机制，为卵巢癌的预后判断及个体化治疗提供基于lncRNA的新靶点。

申请人前期通过芯片在卵巢癌中原创性鉴定了一个新长链非编码RNA(lncRNA-TC0101441)，但TC0101441在卵巢癌转移中的作用环节及分子机制尚未阐明。因此，本项目从临床、细胞、动物水平展开了一系列实验研究，结果如下：①临床层面，发现TC0101441在卵巢癌组织中的表达明显高于正常卵巢上皮组织，TC0101441的高表达与卵巢癌的恶性生物学特征，包括FIGO分期（Ⅲ-Ⅳ期）、组织学分级（G3）、淋巴结转移密切相关。单因素及多因素生存预后分析显示TC0101441是卵巢癌总生存期及无进展生存期的独立预后因素。②体外细胞层面，通过一系列功能缺失及获得实验,肿瘤转移基因芯片、鬼笔环肽细胞骨架、迁移侵袭实验，阐明TC0101441能诱导EMT促进卵巢癌细胞的迁移侵袭。③动物层面，活体成像及大体解剖结果均表明TC0101441能诱导EMT促进卵巢癌裸鼠腹腔种植瘤的播散转移。④在机制探索方面，借助生物信息学及UCSC基因组数据库（Hg19）分析发现TC0101441反义链临近50kb序列范围内存在肿瘤转移相关基因KiSS1。后续验证发现敲低/过表达TC0101441显著改变KiSS1的表达，提示TC0101441可能通过上调临近靶基因KiSS1发挥功能。进一步借助rescue补救实验，通过鬼笔环肽细胞骨架及Transwell侵袭实验，发现TC0101441诱导EMT促进卵巢癌细胞的侵袭转移依赖于KiSS1，提示TC0101441 可通过“TC0101441-KiSS1-EMT途径”促进卵巢癌的转移。这些结果不仅有利于全面理解 TC0101441促进卵巢癌侵袭转移的作用环节及分子机制，而且为卵巢癌的预后判断及个体化治疗提供基于lncRNA靶点的新思路。