长链非编码RNA SNHG3和CEBPZ-AS A-to-I过编辑促进肺癌的发生、发展及其机制

Since the depth analysis of high-throughput sequencing data indicating that the adenosine to inosine (A-to-I) RNA editing is a common genetic modification, it has become a new hotspot in cancer research. The A-to-I editing can alter base sequence of host long non-coding RNA (lncRNA), which may influence the regulation of target coding genes. We have analyzed transcriptome data of lung cancer and found that lung cancer developed dysregulated editing hotspots and generally showed hyper-editing level. Based on the TCGA data, we further revealed three aberrant editing sites, A1746I and A1075/1126I that are respectively located in lncRNA SNHG3 and CEBPZ-AS to be significantly associated with poor lung cancer prognosis. Moreover, the bioinformatics analysis indicated that these A-to-I sites may alter structural stability of their host lncRNAs or the interaction between lncRNAs and RNA binding protein as well as microRNAs. Thus, we hypothesized that the high editing level of SNHG3 A1746I and CEBPZ-AS A1075/1126I can promote the development of lung cancer via interruption on function of their hosts. Here, this project intends to investigate the editing levels of above three A-to-I hotspots in clinical lung cancer samples and analysis their associations with the onset and prognosis of the disease using the BASEscope technology; To reveal the molecular mechanism underlying the biological effects of the A-to-I sites on lung cancer through a series of in vivo and in vitro experiments, which will be conducted on high editing pattern lung cancer cell lines that are constructed using the RNA editing for programmable A to I replacement system. The results from this project will provide effectively scientific evidences for molecular targeted therapy of lung cancer.

高通量测序数据的深度解析表明腺苷至次黄苷碱基（A-to-I）RNA编辑是常见的遗传饰变，成为肿瘤研究的新热点。A-to-I编辑会引起lncRNA碱基序列改变，极可能影响其对靶基因的调控。课题组前期解析转录组数据揭示肺癌具有异常的编辑热点且存在过编辑现象；分析TCGA数据发现lncRNA SNHG3的A1746I和CEBPZ-AS的A1075/1126I高编辑与肺癌预后差有关。生物信息学预测上述三个位点可分别干扰其lncRNA结构稳定性或与RNA结合蛋白、微小RNA的结合，故推测上述A-to-I过编辑可影响lncRNA功能而促进肺癌的发生发展。本项目拟利用BASEscope技术检测临床样本上述3个A-to-I编辑的水平及其与肺癌发病和预后的关联；应用RNA编辑技术构建肺癌高编辑模式细胞系，通过系统的体内、体外研究，揭示A-to-I过编辑影响肺癌的生物学机制，为肺癌的分子靶向治疗提供科学依据。

腺苷-肌苷RNA编辑是肿瘤新的生物标志物类型，但目前对其在肺癌发生发展中的作用知之甚少。本项目在前期分析8对肺癌癌组织与癌旁正常组织二代测序数据和TCGA肺癌腺苷-肌苷编辑数据的基础上，旨在分析SNHG3 c.1746A>I和CEBPZOS c.1122A>I、c.1075A>I三个腺苷-肌苷编辑位点与我国人群肺癌发生发展的关联及其机制。项目在广州地区收集了262例肺癌患者的临床信息及相应的肺癌组织与癌旁正常组织，并成功获取154例肺癌患者完整的生存信息。在上述样本中检测上述三个位点的编辑水平，发现相较于癌旁正常组织，SNHG3 c.1746A>I和CEBPZOS c.1122A>I在癌组织中均存在过编辑现象，且c.1746A>I在有淋巴结转移和远端转移者的编辑水平显著高于无转移者，c.1122A>I在高T分期和有淋巴结转移者的编辑水平亦显著高于低T分期和无淋巴结转移者。两位点高编辑患者的总生存期均短于低编辑者，且死亡风险更高。然而，c.1075A>I在癌组织与癌旁正常组织间的编辑水平无显著差异，且与临床分期无关。表型实验显示，编辑型SNHG3对肺癌细胞转移和侵袭能力的促进效应显著强于野生型SNHG3，机制在于c.1746A>I编辑可增强SNHG3与转录因子SSRP1的结合能力，进而上调GSS、PHKG2、CBS、ECHS1、CPT1B、HMMR和THBS3的蛋白表达，这些靶基因可以促进细胞外基质-受体相互作用、脂肪酸代谢和铁死亡抵抗；c.1122A>I编辑可导致CEBPZOS表达下调，进而抑制该基因对肿瘤细胞增殖、克隆形成、转移和侵袭能力的抑制效应，机制在于c.1122A>I可导致CEBPZOS基因3’-非翻译区新增miR-150-5p的结合位点，进而对肿瘤生长因子-β信号等通路分子的表达产生潜在影响。此外，上述两位点还影响肺癌细胞对多个抗肿瘤药物的敏感性。以上结果说明，SNHG3 c.1746A>I和CEBPZOS c.1122A>I可影响宿主基因的功能而发挥促癌效应，有望成为肺癌进展和预后预测的功能性生物标志和治疗的新分子靶标。