ADAR介导CDK13的RNA编辑在食管鳞癌中的预后和分子作用研究

Presently, esophageal cancer is still a disease with high mortality rate, meanwhile esophageal squamous cell carcinoma (ESCC) is the most commonly observed histological subtype of esophageal cancer. RNA editing, which is defined as the nucleotide sequence change of RNA transcripts relative to that of the encoding DNA, could enhance the RNA diversity, and lead to changes in amino acid sequence and alternative splicing, thereby increasing the complexity of gene expression. RNA editing has been connected to cancer development and progression. However, unfortunately, RNA editing has not been well studied in genome-wide in cancers, especially merely documented in esophageal cancer. Through high-throughput sequencing, our preliminary data suggest that specific sites in CDK13 is occurred RNA editing in ESCC tissues, and ADAR which catalyze RNA editing process is high expressed in ESCC tumors. Here, we raise a hypothesis that CDK13 editing in RNA level which is mediated by ADAR over-expression could enhance ESCC development. We plan to use 8 ESCC cell lines to investigate the ADAR and CDK13 editing influencing of tumor cell proliferation, migration, invation, apoptosis and other biological characteristics by the lentiviral-mediated over-expression strategy. Furthermore, we will determine the carcinogenesis of CDK13 in the nude mice tumor model by the high expression their editing-type transcripts. As it was reported that CDK13 interacts with the ASF/SF2-associated protein p32, and involved in splicing regulation. We will test the effect of combination that wild-type and edited CDK13 with p32, and introducing RNA-seq to profile the RNA splicing alteration compare edited CDK13 with its wild form. Finally, this study intends to expand more ESCC clinical specimens (200 ESCC patients) to validate their frequencies, and analyze their correlation with prognosis data and other clinical-pathological information. In conclusion, we devised the workflow of this project by steps that from clinical samples to molecular experimental model to clinical samples, hoping to provide a novel type of diagnosis or prognosis biomarker in ESCC.

目前食管癌的5年生存率较低，诊断和治疗需要从分子水平获得新的思路。RNA编辑是一种特定分子机制下形成的RNA和DNA序列的差异，在前期研究中我们通过“组学”检测发现CDK13的固定位点在食管鳞癌组织中发生特异性的RNA编辑，并与ADAR在食管癌中高表达相关。故我们假设在食管鳞癌中ADAR的表达上调导致RNA编辑的发生并在特定位点受到癌细胞的选择。本项研究将利用慢病毒介导ADAR过表达检测其与CDK13 RNA编辑的关系；检测CDK13的RNA编辑对食管鳞癌细胞增殖、迁移、侵袭、凋亡等生物学特性的影响，并采用裸鼠荷瘤实验做体内的进一步确认；检测编辑型CDK13和野生型与p32结合能力是否变化，并进一步利用高通量测序鉴定编辑前后CDK13诱导的RNA剪切在转录组整体上的变化。最后对上述RNA编辑位点在200例食管鳞癌组织中验证，分析其与预后及各种临床参数的相关性，探索其作为分子标志物的可能。

本项目计划对食管鳞癌（ESCC）中特定的RNA编辑开展研究，即CDK13的RNA编辑在ESCC中的作用，线索来自于我们之前对13对食管鳞癌转录组和外显子组的测序和分析结果。但是，研究过程中未观察到CDK13的RNA编辑对食管鳞癌细胞系的功能有重要影响。故我们在更广范围内对RNA编辑在癌症中的作用和发生的机制进行了探索。第一项研究是I型干扰素介导的ESCC的RNA编辑。首先，我们确认了ADAR1蛋白的表达与ESCC上RNA编辑发生的关系。对ESCC患者组织的转录组分析发现ADAR1的表达量与STAT1、STAT2和IRF9的表达相关。体外实验进一步证实，ESCC细胞内ADAR1的蛋白量与JAK/STAT通路被I型干扰素的活化有关。对I型干扰素处理前后的ESCC细胞系进行转录组测序发现，RNA编辑的数量和编辑型频率都显著提升。这些发现为解释ESCC的发生开辟了新的方向。第二项研究是AZIN1 RNA编辑在非小细胞肺癌中的作用。我们聚焦于AZIN1 (antizyme inhibitor 1)上一个特定的RNA编辑位点S367G。使用了30例NSCLC患者的手术标本和13个相应疾病类型的细胞系，发现AZIN1 S367G的RNA编辑是NSCLC中的普遍现象。我们发现AZIN1 RNA编辑携带的病例中该基因的 表达量上升，同时这些病例中ADAR基因的表达量也有所升高。该基因的RNA编辑导致蛋白从细胞膜到细胞核定位的改变，并与癌细胞增殖、浸润、迁移等一系列能力的提升相关。对AZIN1 RNA编辑功能的抑制可能为肺细胞肺癌的治疗提供新的途径。