结直肠癌中的RNA质量监控基因upf1突变及其分子机制研究

Colorectal cancer(CRC) is one of the common malignant cancers in the world, its incidence and mortality have been increased in china this years. Genetic instability in CRCs leads to gene mutation, frameshift, deletion and insertion, which should have been supervised and repaired by multiple cellular mechanisms. Nonsense-mediated mRNA decay(NMD) pathway is a post-transcriptional regulator of gene expression affecting above 10 % of all mRNAs by mRNA degradation, and RNA surveillance gene upf1(regulator of nonsense transcripts 1) is a key component of the pathway. Studies show that some substrates of NMD pathway ‘escaped’ from degradation in CRC cells and tissue，which indicate NMD pathway deficiency in the tumor. In this project, we focus on the NMD pathway in CRCs tissues and discovered intronic mutations in upf1 gene with abnormal protein expression. To figure out whether the upf1 intronic mutation indeed disturbed the function of protein, we intend to imitate the mutations in vitro and trace the splicing of upf1 gene. Meanwhile, this project will explore how the NMD pathway regulate expression of apc (adenomatous polyposis coli) mRNA, a tumor-suppressor gene in CRCs, at post-transcriptional level selectively. Further study will explore upf1 deficiency in CRC cells by detecting different expression of NMD substrates, along with monitoring proliferation, apoptosis and inflammatory response of the CRC cells. Through crosstalk between upf1 mutations and expression of NMD substrates such as apc mRNA, we could try to investigate how the dysregulations of gene expression interfere activity of CRCs. Along with durative sequencing on upf1 gene, we will illustrate the spectrum of upf1-NMD pathway deficiency in CRCs. By evaluating this malignant cancer through the gene expression regulation and RNA surveillance pathway, this project could initiate a new perspective on molecular mechanisms research for CRC diagnosis and therapy.

结直肠癌CRC这一常见恶性肿瘤在中国的发病率与致死率逐步提高，其遗传物质的不稳定导致了基因突变、移码、缺失和插入，细胞应对进行监督修复。无义介导mRNA降解NMD途径是重要的转录后基因调控者，对10%以上mRNA进行监督降解，RNA质量监控基因upf1是其关键因子。研究显示CRC中部分NMD底物“逃脱”降解，暗示了NMD途径缺陷。本项目检测CRC中NMD途径，发现upf1基因内含子突变伴随蛋白异常表达。研究计划在体外模拟突变并追踪基因剪接行为，探索突变对蛋白的影响。此外，本项目将分析NMD途径对抑癌基因apc的转录后调控，并检测upf1缺陷对CRC中NMD底物的影响，同时监测细胞增殖、凋亡和炎性反应。通过关联upf1突变和apc等基因，我们将分表达失调对肿瘤细胞活动的干扰，并绘制upf1-NMD缺陷图谱；从基因表达调控和质量监控的角度研究疾病，将为CRC诊断治疗分子机制研究提供新视角。

结直肠癌（colorectal cancer，CRC）是消化道常见的恶性肿瘤，其病因和发病机制十分复杂。腺瘤性结肠息肉病基因（adenomatous polyposis coli，APC）与结直肠癌密切相关，是该恶性肿瘤的关键抑癌因子。无义突变介导的mRNA降解途径（Nonsense-mediated mRNA decay，NMD）缺陷会导致癌基因和抑癌基因异常表达，促进肿瘤发生发展。本研究通过探索NMD途径对抑癌基因APC的调控，为结直肠癌的分子机制研究和防治策略提供新思路。我们通过测序分析发现结直肠癌样本中存在UPF1内含子突变、截短版本表达和APC基因选择性剪接体异常。功能分析显示UPF1内含子突变对其所临近内含子的剪接行为无明显作用，而UPF1基因的截短版本则显著导致了其蛋白功能损伤，UPF1缺陷被证实参与调节APC选择性剪接，并通过这一调节作用对结直肠癌细胞迁移及凋亡相关。延展的数据库分析提示UPF1基因表达还能从干扰炎性细胞浸润等多方面影响结直肠癌进展。以上结果说明结直肠癌中UPF1基因缺陷导致了APC基因选择性剪接异常，并参与结直肠癌发展。