Alu序列的indel遗传变异影响circRNA0050386剪接在肺癌发生发展的作用及机制研究

circRNAs are important molecules that help to exactly regulate the function of coding-genes. The flanking Alu repeat sequences are crucially important for the circularization of circRNAs. Structural variations in the Alu sequence may interfere with circRNA formation, and thus affect the regulation of downstream genes. Our preliminary study indicated that the expression of circRNA0050386 in lung cancer tissues and plasma were significantly lower than those of normal controls, and the expression level of circRNA was positively correlated with SRGAP3 gene's expression. There are genetic variants in the flanking Alu sequence of circRNA0050386. Among of them, c.536-/TTTA indel had a significant correlation with lung cancer risk and with circRNA0050386 expression. Thus, we hypothesized that c.536-/TTTA is associated with lung cancer risk via its effect on circRNA0050386 splicing, thereby interfering with the regulation of target genes. Here, in this project we plan to test the association between the c.536-/TTTA and lung cancer risk as well as prognosis by genotyping the indel and detecting the circRNA expression in a two-center large population study. To investigate the molecular mechanism of the c.536-/TTTA in lung cancer development and progression, a series of human bronchial epithetical model cell lines carrying different genotype of the c.536-/TTTA will be constructed via CRISPR/Cas9 technique, and a series of functional assays will be further implemented in vitro and in vivo. This project will help to clarify the mechanism of lung cancer pathology, and provide scientific evidences for the prevention and treatment of lung cancer.

circRNA是精细调控编码基因功能的重要分子，其侧翼Alu重复序列对circRNA成环有着至关重要的作用，该区域的结构变异极可能会干扰其形成，影响对下游基因的调控。课题组前期研究发现circRNA0050386在肺癌组织和血浆中的表达显著低于正常对照，且与抑癌基因SRGAP3表达呈正相关。该circRNA Alu序列存在多个变异位点，预实验发现c.536-/TTTA indel影响其表达并与肺癌发病风险有关，据此我们假设：该indel变异可能通过影响circRNA剪接，进而干扰其调控靶基因的效应而影响肺癌的发生发展。本项目拟在双中心大样本人群检测该indel的频率分布及circRNA的差异表达，分析其与肺癌发病的关联；通过基因编辑技术构建一系列含不同indel基因型的肺模式细胞系，整合体内、体外等实验方法系统研究该indel影响肺癌发生发展的分子机制，为肺癌的高危人群筛选和防治提供依据。

环状RNA（circRNA）是一类新型非编码RNA（ncRNA），在作为新型的疾病生物标志方面具有明显的优势和应用前景。circRNA侧翼Alu重复序列对circRNA的形成至关重要。本研究旨在探索circRNA0050386侧翼Alu 序列的c.536-/TTTA遗传变异对circRNA形成和表达的影响及揭示其生物学机制。项目在广州和苏州两地区人群中揭示了c.536-/TTTA遗传变异与我国人群肺癌发病存在关联性。circRNA0050386具有癌组织低表达特异性，且与c.536-/TTTA遗传变异相关。系列功能实验显示，circRNA0050386能够抑制肺癌细胞的增殖、克隆形成、裸鼠成瘤以及迁移侵袭等肿瘤生物学能力。机制实验显示，circRNA0050386通过与SRSF3蛋白结合，下调SRSF3蛋白表达水平，进而下调FN1的表达，抑制肺癌细胞的生长及转移。以上结果说明，c.536-/TTTA遗传变异可通过影响circRNA0050386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。circRNA0050386发挥抑癌作用，可作为肺癌治疗的潜在新分子靶标。