一个功能未知的环状RNA circ-0005615调控结直肠癌侵袭转移的机制研究

Using microarray analysis and quantitative real-time PCR (Q-RT-PCR), we identified a novel circular RNA (circRNA), circ-0005615 (CR), with unknown function that is highly expressed in colorectal cancer (CRC) tissues. Our previous work has demonstrated that CR is significantly overexpressed in CRC tissues, and positively correlated with TNM stage, negatively correlated with differentiation and prognosis. Knockdown of CR significantly inhibited migration and invasion of CRC cells. Based on preliminary experiment and bioinformatics analysis, we found that CR could act as competitive endogenous RNA (ceRNA) to promote the invasion and metastasis of CRC via absorbing miR-519 and up-regulating HuR; which was also our hypothesis. We plan to validate the hypothesis by the manners of RNA immunoprecipitation, RNA pull down assays, and dual-luciferase reporter assay system, rescue experiment designation to underlying the molecular mechanism in vivo and in vitro. Thus we can carry on a clinical assessment of the relationship between the ceRNA networks of CR/miR-519/HuR and the invasion and metastasis of CRC. Up to now, there is no information about how CR is working in CRC, therefore our study would offer a new therapeutic target for the diagnosis and treatment of CRC.

circ-0005615（简称CR）是前期应用高通量芯片筛选出的一条在人结直肠癌（简称肠癌）组织中差异高表达、功能未知的环状RNA。验证发现CR在肠癌中平均高表达5倍以上，与分期正相关、与分化程度及预后负相关；沉默CR可显著抑制肠癌细胞株侵袭迁移。预实验及生物信息学分析提示CR可吸附“抑癌基因”miR-519，并上调促转移基因HuR表达。故我们提出“CR通过竞争性内源RNA（ceRNA）机制吸附miR-519上调HuR表达，从而促进肠癌侵袭转移”的科学假说。本研究拟体内、体外实验明确CR促进肠癌侵袭转移的生物学功能；采用RIP、RNA pull-down及双荧光素酶报告基因等技术，设计拯救实验，阐明CR通过ceRNA机制促进肠癌侵袭转移的分子机制；临床评价CR/miR-519/HuR的ceRNA网络与肠癌侵袭转移的相关性。关于CR在肠癌中的功能机制尚无报道，本研究可为肠癌防治提供新靶标。

环状RNA（circRNAs）是由外显子前体mRNA反向剪接产生的非编码rna，在肿瘤中具有多种作用。然而，在结直肠癌中，许多circRNAs的作用仍不清楚。在本研究中，我们通过高通量芯片分析了5对结直肠癌患者样本中circRNA的表达谱，并发现在结直肠癌组织中，一个circRNA，hsa_circ_0005615（circ5615）显著上调。circ5615来自NFATC3的二号外显子，其上调与大肠癌患者更高T分期和更差预后密切相关。体外和体内研究表明，结直肠癌细胞系中circ5615基因的敲除抑制了肿瘤细胞增殖和阻滞细胞周期，而过表达促进了肿瘤细胞的恶性表型。机制方面，RNA免疫共沉淀、生物素偶联探针下拉和荧光素酶报告分析显示circ5615与miR-149-5p结合，可能起到类似miR-149-5p海绵的作用。此外，通过RNA-seq和生物信息学分析，确定β-catenin稳定性调节因子tankyrase（TNKS）是circ5615下游蛋白，是潜在的miR-149-5p靶点。我们进一步证实了circ5615诱导β-catenin和cyclin D1的上调。结果表明，circ5615具有与miR-149-5p竞争通过竞争性内源RNA机制促进TNKS表达，激活Wnt/β-catenin通路在结直肠癌中发挥致癌作用。