以翻译起始因子eIF3为靶点系统鉴定与验证食管癌中具有潜在编码功能的非编码RNA

Non-coding RNAs (ncRNAs) play very important role in the biological processes. There is evidence that some previously supposed ncRNA is able to produce functional protein molecules published in journal of Cell and journal of Nature in 2005, which overthrew the traditional conclusion of ncRNAs could not be translated to functional protein. Currently a big ban on understanding the role of ncRNA is that there is not a systematic method to accurately predict the ncRNAs, while ribosome profiling and mass spectrum have been applied to solve it. Based on the review of literatures and previous work, we found that translation initiation factor eIF3b could capture the RNAs which were initiating ribosome assembly to produce proteins. In this project, we will mainly adopt CLIP-seq technique with eIF3b as the target molecule to identify the difference in the characteristics between coding RNA and ncRNA in esophageal cancer cells and normal esophageal epithelium cell. We will also combine the data from CLIP-seq and CAGE-seq, RNA-seq and mass spectrum to establish a predict model to differentiate the ncRNAs with potential protein-coding function from those currently thought to be without coding function. This model will help to illustrate the role of eIF3b as a marker to indicate the coding function of certain RNA candidates, update the current ncRNA annotation criteria and relative ncRNA database. Moreover, results from this project will potentially recover the role of new ncRNA with RNA coding function in esophageal cancer occurrence and development. It will have certain instructive meaning in ncRNA research of the other diseases and other species.

非编码RNA(ncRNA)在真核细胞中发挥重要作用。2015年《cell》、《Nature》发表的文章颠覆了ncRNA不能编码功能性蛋白的传统认识。现有基于核糖体展示和质谱技术的研究结果在回答哪些RNA可以编码及其详细特征方面存在明显不足，经过文献分析和预实验我们发现以翻译起始因子eIF3b为靶分子能最大程度地捕获正在启动翻译核糖体组装的RNA。本课题拟以人食管癌细胞为模型，eIF3b为切入点，采用CLIP-seq技术、5’端帽测序、转录组测序联合质谱分析探索具有潜在编码能力的ncRNA，并进行蛋白表达验证和生物学功能鉴定。本课题的潜在研究结果不但能在全细胞转录组水平上回答哪些ncRNA能编码及其可能的鉴别特征，并能推动现有ncRNA数据库和注释规则的升级，还可能发现新的具有编码能力的ncRNA与食管癌发生发展的关系。本课题的研究策略与结果对其他疾病及物种的ncRNA研究也将具有借鉴意义。

长链非编码RNA(Long non-coding RNA,LncRNA)可在细胞生物学的多个水平上发挥作用,形成复杂的分子调控网络,直接或间接调节多种生物学过程,其异常表达也与肿瘤的增殖、侵袭及迁移密切相关。近年来研究显示LncRNA不仅仅以RNA的形式发挥功能,部分LncRNA是可以编码功能性多肽(hidden polypeptides或者micro-peptides)的,如影响肿瘤细胞的克隆形成、EMT转化等能力。但目前基于Ribo-seq、RNC-seq和质谱技术的研究结果在回答哪些RNA可以编码及其详细特征方面仍存在明显不足。为了筛选与食管癌相关的编码性LncRNA，本研究把翻译起始复合物重要组件eIF3b作为靶分子，利用CLIP-seq富集食管癌细胞系中处于翻译过程当中的转录本。之后利用iTRAQ质谱技术检测与CLIP-seq实验同期的食管癌细胞系中的蛋白；整合分析CLIP-seq数据和iTRAQ质谱数据，批量筛选具有潜在编码性的LncRNA，并探究其与食管癌恶行表型的关系。.研究结果表明食管癌中存在着许多具有潜在编码性的LncRNA，eIF3b能够以序列特异性的方式有效富集具有潜在编码性的LncRNA；本项目筛选186个具有潜在编码性的LncRNA，与已知蛋白相比,由LncRNA编码的多肽具有低覆盖率和低丰度的特性；LncRNA AC027682.1可以编码15KD的多肽CTCF-AS，该多肽可以促进食管癌细胞的增殖，增强食管癌细胞的克隆形成和划痕愈合能力。CTCF-AS在食管癌在内的多种癌组织中高表达，且高表达CTCF-AS的食管癌患者生存显著较差。 .本项目基于高通量的测序数据与质谱数据的客观对应关系在全转录组层次鉴定具有潜在编码性的LncRNA，并总结这类LncRNA的特征，以完善人们对LncRNA的认识，推动现有LncRNA 数据库资源的升级。本项目的研究结果将对本领域研究产生非常有价值的基础信息和拓展信息。