NEIL1基因RNA编辑在结直肠癌侵袭转移中的分子功能与作用机制研究

RNA editing is defined as post-transcriptional alteration of RNA sequences. Previous studies have shown that RNA editing is closely associated with the development and/or progression of human cancers. Recently, we found a higher frequent editing event within the transcript of NEIL1 gene in advanced colorectal carcinoma (CRC) tissues with liver metastasis compared to that in early stage CRC tissues using next-generation sequencing. However, the functions and mechanisms of NEIL1 editing in the invasion and metastatic process of CRC have not been evaluated.. In this project, firstly, editing type NEIL1 expression vector will be transfected into the CRC cells to assess the effect of Edit-NEIL1 on the invasive and metastatic behavior of CRC cells. Next, gene expression microarray and ChIP-seq will be applied to screen the direct downstream targets of Edit-NEIL1 in CRC cells and the results will be verified. Furthermore, the editing rate of NEIL1 and its target genes expression in CRC tissues will be tested by pyrosequencing and immunohistochemistry respectively, and the association between the editing rate of NEIL1 and expression of target genes will be assessed accordingly. Loss or gain of function in vitro assay will be employed to identify the critical target genes modulating by Edit-NEIL1 via transfecting Edit-NEIL1 overexpression cells with gene overexpression vector or siRNA targeting correlative genes. Finally, the mechanisms of Edit-NEIL1 regulating its critical target will be explored through luciferase reporter gene array and EMSA. This project will provide new clues to clarify the metastatic mechanisms of CRC.

RNA编辑是基因转录后水平RNA序列改变的分子事件，与肿瘤发生发展相关。最近我们通过二代测序在肝转移的晚期结直肠癌（CRC）中发现NEIL1基因存在高编辑率。但编辑型（Edit）NEIL1在CRC侵袭转移中的分子功能与机制不清楚。.本项目拟首先探讨过表达Edit-NEIL1基因对CRC细胞侵袭和转移等生物学行为的影响；通过表达谱芯片和ChIP-seq技术筛选Edit-NEIL1直接作用和调控的靶基因；并在大病例CRC组织中检测和分析NEIL1基因编辑与靶基因表达的相关性和临床肿瘤学意义。进一步，在过表达Edit-NEIL1的细胞中, 通过基因回复（沉默或导入）实验，观察靶基因回复后对CRC细胞侵袭和转移的影响，从而锁定Edit-NEIL1作用的关键靶基因和相关信号通路；并在荧光素酶报告基因和EMSA等实验中确证Edit-NEIL1对靶基因的调控机理。本研究将为揭示CRC转移机制提供新线索。

研究发现RNA序列改变即RNA编辑与肿瘤发生发展密切相关。我们通过二代测序在肝转移的晚期结直肠癌（Colorectal cancer, CRC）中发现NEIL1基因存在RNA编辑。但野生型（WT）及编辑型（Edit）NEIL1在CRC侵袭转移中的分子功能与机制不清楚。进一步我们明确NEIL1基因在CRC中确实存在RNA编辑，且WT-NEIL1蛋白在进展期的CRC中存在异常高表达。NEIL1蛋白表达水平与患者预后显著负相关。通过构建稳定过表达及用CRISPR/Cas9方法稳定敲除人或小鼠结直肠癌细胞内的NEIL1基因细胞株，明确了WT及Edit-NEIL1过表达可显著促进CRC细胞的干细胞特性、成瘤及转移的功能。机制方面发现NEIL1通过促进STAT3蛋白稳定增强CCL2、 GM-CSF、IL8、CXCL1等多种细胞因子分泌，导致肿瘤组织内免疫抑制微环境形成及CD8+T细胞减少，并最终促进CRC恶性进展。此研究为CRC提供了新的治疗靶点及联合肿瘤免疫治疗的实验基础。