基于RNA-seq策略研究天然反义转录本的调控机制

Natural antisense transcripts (NATs) are coding or non-coding RNAs with sequence complementarity to other transcripts (sense transcripts). Natural antisense transcripts have already been found to function at several levels of eukaryotic gene regulation including translational regulation, RNA stability, alternative splicing, trafficking and genomic imprinting. Changes in antisense transcription have been implicated in pathogenesis, such as cancer or neurological disease. These RNAs could potentially regulate the expression of their sense partner(s) at either the transcriptional or post-transcriptional level through a variety of biological mechanisms, such as transcription interference, RNA masking, dsRNA-dependent mechanisms and directed remodeling of chromatin at target loci. While the majority of studies have focused on the mapping and evolutionary aspect of NATs and SAS pairs, only a few studies have interrogated and validated their abundance in different human tissue.There are still huge gaps in our understanding of NATs of mechanistic basis for their functions in cancer cell. .Our aim is to investigate comprehensively the differential expression levels of SAS pairs between the normal and cancer cell based strand-specific massively parallel cDNA sequencing (RNA-seq) method. Strand-specific RNA-seq is a powerful tool for transcript discovery, genome annotation and expression profiling. First, we will build the global gene expression profile for the normal and cancer cell. Second, we will identify antisense transcript displaying a high level of expression specifically in normal and cancer tissues respectively. Furthermore, the genome-wide analysis of differences will be performed in antisense and sense transcriptomes between normal and cancer samples. Third, we will analyze the functional role of these antisense transcript in cancer cell with combining bioinformatics methods. The orientation specific qPCR of selected SAS pairs will be used to validate their expression in several cancer cell lines. We hope this research can provide insights into the biological mechanisms underlying altered antisense-to-sense levels and cancer treatment.

天然反义转录本(NATs)是由蛋白编码基因的反义链转录产生，属于一种特殊类型的非编码RNA, NATs具有多层次的调控活性，比如转录后调控、剪切调控和转运调控等，目前对于NATs的组成、功能和调控机制等方面的理解还有很大差距，因此亟需发展和应用深度测序技术揭示NATs的内在本质和调控机制。围绕上述科学问题，本项目拟以5种生殖相关的癌症细胞和3种正常细胞(卵巢细胞、乳腺细胞和乳腺上皮细胞)为研究对象，通过链特异性的RNA-seq方法构建癌症细胞和正常细胞的全景表达谱，研究天然反义RNA在肿瘤细胞发生过程中的作用，建立一套新的天然反义RNA的功能研究体系。最终目的是发现和鉴别一批与癌症相关的NATs，揭示其与肿瘤发生的关系及在不同肿瘤中的表达特征，阐明肿瘤发生过程中NATs的调控机制。发现以RNA分子为对象的治疗靶标和早期诊断标识，为肿瘤等重大疾病的防治提供新的思路和方法。

在本研究中，我们通过链特异性RNA-seq方法获得卵巢癌组织的转录本深度测序表达谱，并比较与传统RNA-seq方法之间差异性，发现ploy(A) RNAs富集方法适合于只关注含ploy(A)尾的转录本研究，并且可以节省测序成本，而链特异性rmRNA-seq方法则更适合于分析和鉴定天然反义转录本（Natural Antisense Transcripts，NATs）并且展现了一个更全面的真核转录表达谱；建立了NATs数据分析模型和表达模式分析模型，通过转录组学方法大规模鉴定四种宫颈癌细胞系中新的NATs和Sense转录本对，并发现不同细胞系有其特定的表达转录本，详细分析鉴定出一些关键的转录本，并发现这些NATs在宫颈癌细胞肿瘤发生的表达调控过程中发挥重要作用，初步探索了天然反义转录本的调控机制。完成对癌症细胞系的基因表达调控模式对比分析，表明癌症细胞具有局部共性的调控表达模式，形成癌症细胞特异表达标签特征，并形成了转录组数据库和在线分析平台。