长链非编码RNA FENDRR拷贝数变异调控Snail1基因影响人群肺癌发病和预后的研究

Copy number variation (CNV) is an important biomarker for disease susceptibility. By using the array-based comparative genomic hybridization (aCGH) platform, we have previously identified a hotspot of CNVs at the chromosome 16q24.1 in lung cancer cases. Fine mapping of this region further revealed the CNV-30795 to be associated with lung cancer risk, which is located in the fourth exon of the long non-coding RNA (LncRNA) FENDRR. Preliminary experiments showed that the CNV can cause a down expression of FENDRR and is associated with up expression of the EMT regulator Snail1. Further bioinformatics analysis shown that there are binding site of FENDRR at the promoter region of Snail1. Thus, we hypothesized that the CNV of FENDRR is associated with lung cancer risk and prognosis via its effect on FENDRR as well as Snail1 expression. Here, this project intends to test the association between the CNV-30795 and lung cancer risk as well as prognosis by genotyping the CNV and detecting the LncRNA expression in a two-center and large population; to investigate the molecular mechanism of the CNV-30795 on regulating the expression of FENDRR and Snai1 by constructing a serious of human bronchial epithetical model cell lines carrying different copy number of the CNV-30795 via TALEN technique. Finally, a series functional assays will be used to illustrate the biological mechanism of CNV-30795 on modulating LncRNA FENDRR and Snail1 gene in lung cancer development. This project will help to clarify the mechanism of lung cancer pathology, and provide scientific evidences for prevention and treatment of lung cancer.

拷贝数变异(CNV)是影响疾病易感性的重要生物标志物。预实验检测了全基因组染色体变异位点，发现肺癌病例在16q24.1区域存在拷贝数变异热点；精细定位发现CNV-30795可能与肺癌发病存在关联，它位于长链非编码RNA(LncRNA) FENDRR第四外显子，可降低该LncRNA的表达，并与EMT调控因子Snail1基因高表达有关。生物信息分析发现Snail1基因启动子存在该LncRNA的结合序列，故推测FENDRR的拷贝数变异可降低其LncRNA表达并通过调控Snail1基因而影响肺癌的发生发展。本项目拟在两中心、大样本人群检测上述拷贝数变异和LncRNA表达，探讨其与肺癌发病和预后的关联；应用TALEN技术构建系列含拷贝数变异的肺癌模式细胞系，系统地运用多种功能实验方法探讨该CNV调控LncRNA及Snail1的机制，揭示FENDRR拷贝数变异的生物学功能，为肺癌防治提供科学依据。

“遗传性缺失”是后GWAS时代遗传易感性应用于肿瘤防治的主要挑战。基于拷贝数变异（copy number variation，CNV）在肿瘤遗传易感性中的重要意义，本研究旨在预试验CNV芯片筛查结果的基础上，分析了FENDRR拷贝数变异CNV-30795与我国人群肺癌发生发展的关联及其生物学机制。项目在广州和苏州两地区收集原发性肺癌和按性别相同、年龄一致匹配的健康对照样本并完成了样本DNA的提取，在共计2072例病例和2077例对照中检测了CNV-30795的拷贝数，发现≥3-copy基因型携带者发生肺癌的风险比2-copy常见基因型携带者增加了76%（OR= 1.76，95%CI= 1.46-2.11），但未发现该CNV与环境暴露因素在肺癌发病风险上存在显著的交互效应，亦未发现其与肺癌患者预后和临床进展指标存在显著关联。进一步拓展分析该CNV与肺癌的一个潜在中间表型慢阻肺（COPD）的关系，通过检测1390例COPD病例和1449例肺功能正常对照，发现≥3-copy亦显著增加我国人群COPD的发病风险（OR=1.98，95%CI=1.57-2.51）。系列功能实验显示，CNV-30795与FENDRR表达呈显著负相关，其拷贝数增加所形成的新截短FENDRR启动子可能与原FENDRR启动子区竞争性结合EGR1和TFAP2A等转录因子，而降低FENDRR的表达。FENDRR在肺癌组织中的表达低下，其过表达可显著抑制肺癌细胞的增殖、克隆形成、裸鼠成瘤能力，促进细胞凋亡并影响细胞周期，但不影响细胞转移和侵袭能力。表达谱芯片显示FENDRR可调控多个蛋白编码基因的表达，其中其可通过竞争性结合miR-424而促进抑癌基因FOXF1的表达。然而，FENDRR与Snai1表达无显著关联。以上结果说明，CNV-30795可通过影响类抑癌基因FENDRR而影响我国人群肺癌的发病风险，是我国人群肺癌易感性评估和高危人群筛选的一个潜在标志。FENDRR发挥抑癌作用，可作为一个肺癌治疗的新分子靶标。