环状RNA circITGA7调控结直肠癌关键信号通路RAS及其作用的分子机制

Abnormal activation of Ras signaling pathway is one of the core mechanisms in the development of colorectal cancer.As the molecular switch of cancer, to explore the regulation mechanism of Ras is of great significance. In our previous work,we found a novel circular RNAcircITGA7 significantly down-regulated in colorectal cancer tissues and cells, and it could inhibit the growth and metastasis of colorectal cancer cells in vitro and in vivo.Through the transcriptome sequencing, dual lucifeRase report experiments, RNA pulldown and mass spectrometry analysis, we preliminarily found that circITGA7 could inhibit the growth and metastasis of colorectal cancer via modulating the Ras pathway bysponging microRNAs and binding to coding proteins. In the project, we intends to further clarify the expression and clinical signifiance of circITGA7 in colorectal cancer,and investigatethe molecular mechanism and the role of circITGA7in regulating the Ras signaling pathway by performing CRISPRCas9, RIP, FISHco-localization, and biotinylated miRNA pulldown assays. Furthermore, the regulation mechanism of downregulated circITGA7 expression in epigenetics, transcription and splicing will be also studied in theresearch. The study will clarify the mechanisms of circular RNA in the regulation of colorectal cancer growth and metastasis, and provide a new approach for the diagnosis, treatment and new drug development of colorectal cancer.

Ras作为癌症的分子开关，是结直肠癌发生发展的核心机制之一，但其调控机制尚不明。我们前期工作发现一种新的环状RNA circITGA7在结直肠癌组织和细胞中表达明显下调，在体内外均能抑制结直肠癌细胞的生长及转移。通过转录组测序、双荧光素酶报告实验、RNA pulldown及质谱分析，我们初步发现circITGA7能靶向吸附miRNA及结合编码蛋白调控Ras通路进而抑制结直肠癌的生长及迁移。本项目拟进一步明确circITGA7在结直肠癌的表达及其临床价值；通过CRISPR Cas9基因编辑技术、RIP、FISH共定位、miRNA pulldown等进一步阐明circITGA7在调控Ras信号通路中的分子机制及其作用，并在表观遗传学,转录及剪切水平探讨circITGA7表达下调的机制。本项目可进一步阐明环状RNA调控结直肠癌生长及转移的机制，为结直肠癌的诊断、治疗与新药研发提供新的路径。

越来越多的研究表明环状RNA在多种肿瘤中均存在异常表达并发挥了关键作用，但环状RNA在肿瘤中的功能和机制大部分仍然是未知的。本课题主要研究了一个新发现的环状RNA circITGA7在人结直肠癌中的表达、功能及分子机制，并初步探讨了其表达调控机制。利用PCR扩增、Sanger测序、RNA酶R消化及放线菌素D处理等技术鉴定了新的环状RNA circITGA7，检测了circITGA7在结直肠癌组织及多种细胞株中的表达，证明了在结直肠癌组织中circITGA7表达下调并与生长、转移呈负相关。成功构建了稳定过表达和干扰细胞株，利用MTT、平板克隆、Transwell迁移及侵袭实验、流式细胞术、裸鼠皮下成瘤实验、脾脏注射肝转移及盲肠原位种植转移模型等实验，发现过表达circITGA7可以抑制结直肠癌的生长和迁移、而干扰circITGA7则可以促进结直肠癌的生长和迁移。通过RNA-seq及western blot实验发现circITGA7调控Ras通路。利用FISH和核浆分离实验证明了circITGA7主要定位于细胞质中。应用生物信息学分析、双荧光素酶报告实验、western blot等实验证明circITGA7通过miRNA-370-3p/NF1信号轴调控Ras信号通路。通过RNA pulldown实验初步证明了circITGA7可以结合蛋白质Vimentin。通过检测circITGA7启动子区甲基化水平、转录因子分析及染色质免疫共沉淀等实验，发现circITGA7的上游调控可能与其甲基化水平、转录因子RREB1和circITGA7上下游内含子相关，而与circITGA7内含子中Alu序列无关。本项目揭示了一个新的环状RNA分子circITGA7在结直肠癌中发挥了重要生物学作用，并初步发现其上下游机制，同时通过ROC相关数据分析发现circITGA7可以作为结直肠癌诊断的潜在分子，并为结直肠癌治疗提供了新的思路和潜在新靶标。本项目已发表论文7篇，其中中科院1区论文3篇，ESI高被引论文1篇，总影响因子达85.04，平均单篇影响因子12.15，被引次数已达178次。已培养硕士研究生4人，博士研究生4人，博士后1人。