FLI1环状RNA在小细胞肺癌发生发展中的作用及分子机理

Small cell lung cancer (SCLC) has the most malignant degree and worst prognosis among lung cancer. We have found that Friend leukemia virus integration 1 （FLI1） is highly expressed in SCLC tissue, and the expression level of FLI1 is positively correlated with the malignant phenotype of SCLC. Meanwhile,we have discovered that there is FLI1 circular RNA (FLI1 circRNA) in SCLC cells, and the circRNA could bind the regulation region of FLI1 promoter. Based on the previous studies, we propose a novel regulatory hypothesis that FLI1 circRNA is invovlved in the function of FLI1 in the carcinogenesis of SCLC. Thus,this project will reveal a novel mechanism of FLI1 circRNA in the regulation of FLI1 and other genes expression through reverse transcription associated trap assay (RAT),deep sequencing, chromosome conformation capture(3C) and chromatin immunoprecipitation(ChIP) etc, in order to clarify the mechanism of FLI1 circRNA invovled in the development of SCLC and to provide the new concept and experimental basis for the study of molecular biomarkers and target therapy in SCLC.

小细胞肺癌（SCLC）是肺癌中恶性程度最高、预后最差的类型。我们前期工作提示FLI1高表达与SCLC恶性表型密切相关，并发现SCLC细胞中存在FLI1环状RNA（circRNA），其能与FLI1等基因启动子调控区相结合，国内外尚未见相关报导。本项目拟基于前期工作基础，探讨FLI1 circRNA参与FLI1促癌调控机制的全新假设，应用逆转录相关捕获法（RAT）、高通量测序、染色质空间构象捕获法（3C）、染色质免疫共沉淀法(ChIP)等技术揭示FLI1 circRNA调控FLI1等基因转录的机制，阐明其在SCLC发生发展中的作用及机理，为SCLC分子标志物及靶向治疗研究提供新的思路与实验基础。

本课题通过启动子dCas9免疫共沉淀分离技术,从FLI1启动子染色质复合体中成功分离鉴定出FLI1外显子环状RNA（FLI1 Exonic CircRNA 1, FECR1）并首次发现FECR1可通过多重表观遗传学机制激活癌基因FLI1：通过与FLI1启动子结合，并募集去甲基化酶TET1，从而促进启动子的DNA去甲基化，同时，通过结合甲基转移酶DNMT1的启动子抑制其表达；通过协调去甲基化酶TET1和甲基化转移酶DNMT1，进而降低DNA甲基化水平并增强FLI1的转录，促进肿瘤转移，实现了对环状RNA功能认知的突破。此外，本项目还发现FLI1基因不仅可以通过编码FLI1蛋白促进肿瘤发生，还可形成FECR1吸附大量miR584-3p，减弱miR584-3p对下游ROCK1基因表达的抑制作用，促进肿瘤的侵袭转移，从而发挥双重促癌作用。更重要的是，我们在小细胞肺癌患者血清的外泌体中鉴定出FECR1，与其他实体肿瘤相比，小细胞肺癌患者血清外泌体中FECR1的表达程度明显升高，且其表达程度与患者的临床分期、化疗疗效及预后等因素密切相关，提示外泌体FECR1可作为一种新型标志物应用于小细胞肺癌的临床监测与预后判断，填补了小细胞肺癌缺乏特异性标志物的空白。