正义lncRNA PET在靶基因翻译过程中的分子调控机制及功能研究

Sense lncRNAs are transcribed from the sense strand of protein-coding genes and contain overlapping sequences with the exons from protein-coding genes. However, the role of sense lncRNAs in regulating gene expression remains largely unknown. Our recent work has identified a previously-uncharacterized sense lncRNA which overlaps with an exon from EphrinA1, a receptor tyrosine kinase ligand. Since this lncRNA enhances the translational efficiency of EphrinA1, we termed it lncRNA PET (lncRNA to promote EphrinA1 translation, PET). PET is upregulated in gastric cancer (GC) tissues and promotes GC cell invasion and metastasis by regulating EphrinA1, which is correlated with distant metastasis and poor prognosis of GC patients. To elucidate the mechanism of PET-mediated EphrinA1 translation, we have discovered another previously-undescribed lncRNA PET-AS ( PET antisense) in the EphrinA1 gene locus, which is complementary to the 3’ end of PET or EphrinA1 mRNA, Further data show that PET-AS directly binds to EphrinA1 mRNA and inhibits its translational efficiency, whereas PET competitively binds to PET-AS and enhances EphrinA1 translation. In this proposal, we will integrate bioinformatics, molecular cell biology, animal models, and clinical pathology to systematically investigate the molecular regulatory mechanism of the PET/PET-AS/EphrinA1 axis in protein translation, and explore the biological function of PET in GC at the molecular, cellular, animal, and clinical levels. This study will contribute to reveal a new strategy mediated by sense lncRNA in genetic information transmission, and provide the experimental and theoretical basis for illustrating the molecular mechanism of how sense lncRNA functions.

正义lncRNA是一类与蛋白质编码基因外显子重叠、并且转录方向相同的lncRNA，然而有关其在基因表达调控中的作用及功能知之甚少。我们发现一个功能未知的、与EphrinA1基因外显子重叠的正义lncRNA，其能增强EphrinA1的翻译效率，并命名为PET（promote EphrinA1 translation）。PET可通过EphrinA1促进胃癌细胞的侵袭和转移。同时，我们还克隆了一个全新的、与PET 3'端互补的lncRNA（PET-AS），其通过与EphrinA1互补结合，抑制EphrinA1的翻译效率；而PET则通过竞争性结合PET-AS促进EphrinA1的翻译。本课题拟在分子、细胞、动物、临床等四个层面，系统研究“PET/PET-AS/EphrinA1”调控轴在靶基因翻译过程中的分子调控机理及其生物学功能。本课题将有助于揭示一类新的由正义lncRNA介导的遗传信息传递方式。

正义lncRNA是指一类与蛋白质编码基因外显子有重叠、转录方向相同的lncRNA。目前，有关正义lncRNA在基因调控过程中的作用及功能知之甚少。我们通过生物信息学技术对GENCODE数据库中所有的人类正义lncRNA及其与蛋白质编码基因的位置关系进行了系统的分析。本项目从一个功能未知的正义lncRNA PET出发，围绕其如何调控靶基因EphrinA1表达及其在肿瘤中的生物学功能等开展了深入研究。我们发现该正义lncRNA PET存在一个反义转录本PET-AS，通过5’和3’-RACE等技术，鉴定并克隆了PET和PET-AS的序列，PET为855nt，PET-AS为2311nt。进一步重点研究了lncRNA PET，PET-AS，EphrinA1 mRNA之间的互作关系。通过检测了mRNA的转录表达、稳定性以及翻译，发现PET和PET-AS主要调控EphrinA1 mRNA的翻译；结合体外RNA pull-down实验和domain mapping实验，发现了“三个碱基互补RNA分子间的相互作用调节靶基因的翻译过程”，——正义lncRNA-PET竞争性地与其对应的反义lncRNA分子PET-AS相互作用，调控靶基因Ephrin A1的翻译过程。有可能揭示一类新的由正义lncRNA介导的遗传信息传递方式。我们进一步系统研究了PET/PET-AS/EphrinA1轴对胃癌细胞恶性生物学行为的调控作用，发现他们PET主要通过增强Ephrin A1表达促进胃癌细胞体外迁移侵袭和体内转移能力，而PET-AS则抑制胃癌细胞迁移侵袭。利用CRISPR技术敲除PET关键区域，显著抑制Ephrin A1翻译表达，并抑制胃癌侵袭转移。我们分析多个胃癌样本队列，发现PET和Ephrin A1在转移性胃癌组织高表达，与胃癌患者不良预后显著相关，可能为胃癌转移的临床诊断和治疗提供新靶点。