环状RNA circIMMP2L促进食管鳞癌恶性进展机制研究

circIMMP2L（简称IM）是前期应用芯片筛选、食管鳞癌（简称EC）临床样本验证获得的功能未知环状RNA。IM在EC癌组织及血浆中均异常高表达，与淋巴结转移和分期显著相关，是预后不良的独立危险因素。体外实验发现沉默IM抑制EC增殖、迁移及侵袭。RNA pull-down、质谱、FISH免疫荧光共定位提示IM特异性结合CtBP1并促其核转位；表达谱芯片筛选结合细胞及临床样本检测提示E-cadherin及P21可能是其下游靶基因。因此提出“IM特异性结合CtBP1，促其核转位招募形成转录抑制复合物抑制下游靶基因E-cadherin及P21表达，促进EC恶性进展”科学假说。本研究拟利用过表达/沉默策略，从体外、体内研究IM在EC中生物学功能；应用RIP、IP、ChIP、设计拯救实验论证其分子机制，并在动物及临床样本层面验证。有关IM促进EC恶性进展机制尚属未知，本研究可为EC诊治提供新思路。

In recent years, circular RNAs (circRNAs) have been revealed to have important roles in tumor progression. As we know that malignant progression is the leading cause of esophageal squamous cell carcinoma (ESCC) death. We identified circIMMP2L (a circRNA derived from exons 2, 3 and 4 of the IMMP2L gene) as a novel circular RNA based on our previous microarray data and the real-time RT-PCR of biobank ESCC tissues. And the biological function of circIMMP2L remained largely unknown. In previous study we found that the expression levels of circIMMP2L in ESCC cancer tissues and plasma were significantly upregulated by comparing with matched normal tissues and plasma of healthy population. Also, the expression level of circIMMP2L was positively correlated with the lymph node metastasis and TNM stage, and negatively correlated with the overall survival time of ESCC patients significantly. ShRNA-mediated silence of circIMMP2L remarkably suppressed the malignant capacity of proliferation, migration, as well as invasion in TE-13 cell line. Furthermore, we identified that circIMMP2L could specifically bind with transcriptional co-repressor CtBP1, which was not a RNA binding protein, by using RNA pull-down, mass spectrometry screening and colocalization analysis by immunofluorescence fluorescence in situ hybridization (IF-FISH). Importantly confocal analysis indicated that circIMMP2L was transferred from the cytoplasm to the nucleus together with CtBP1. Subsequently E-cadherin and P21 were considered as the potential downstream targets of CtBP1 with the combination of microarray screening and the real-time RT-PCR of cell lines and clinical samples. Based on the above preliminary findings, we therefore hypothesized that circIMMP2L could promoting ESCC malignant progression via binding with CtBP1, which was then transferred to the nucleus together with circIMMP2L and recruited a corepressor complex consisting of chromatin modifying enzymes and transcriptional repressors, resulting in the suppression of downstream E-cadherin and P21 expressions. The current project aims to investigate that circIMMP2L could promote ESCC progression both in vitro and in vivo by using silencing and overexpressing strategy at first. Secondly, the above mechanism of circIMMP2L promoting ESCC progression will be deeply validated by the experiments of RIP, IP, ChIP, as well as loss- and gain-of-function studies. Finally, the underlying mechanism between circIMMP2L/CtBP1/E-cadherin and P21 pathway and ESCC progression will be further validated with specimens from in vivo experiments and large number of ESCC patients by chromogenic in situ hybridization (CISH) and immunohistochemistry. To date, no data is available about the function and mechanisms of circIMMP2L promoting ESCC progression. Our study therefore can provide a new idea and approach for the clinical diagnosis and treatment for ESCC.