上皮细胞间质转化相关的lncRNA14功能和机制研究

LncRNAs ( long non-coding RNA), accounting for a major propotion of mamalian transcriptomes, are generally considered as non-protein coding transcripts longer than 200 nucleotides. RNA polymerase II is responsible for the transcription of most lncRNAs. Due to the absence of effective "Open Reading Frame", LncRNAs barely encode any proteins. LncRNAs have been reported to play critical regulatory roles in variety of disease, especially in neurodegenerative disease and cancer. Epithelial-mesenchymal transition (EMT) is an intricate process by which epithelial cells lose their epithelial characteristics and acquire a mesenchymal-like phenotype. Increasing evidences suggest that tumor progresssion is critically involved with the acquisition of an EMT phenotype. LncRNAs have been reported to related to invasion and migration of cancer cells, however few reports have been made for EMT related lncRNAs. We established the TGF beta 1 induced EMT model, which was analyzed subsequenty using lncRNA microarray to screen the EMT related lncRNAs. According to the expression abundance, fold changes and sequence analysis of the promoter regions of these lncRNAs, we selected 23 lncRNAs for real-time PCR verification. In this study, we want to elucidate the functions and specific molecular mechanism of lncRNA 14 on the basis of further bioinformatics analysis. These results will contribute to the establishment of EMT-related lncRNA regulatory network, and thus the identification of key noncoding sequences in EMT, paving the way for finding new targets for tumour diagnosis and therapy.

长链非编码RNA是广泛存在于哺乳动物细胞中的一类非蛋白编码序列，通常由RNA聚合酶II转录形成，长度超过200nt，由于缺少有效开放阅读框，不能编码蛋白。研究发现，lncRNA与一系列重大疾病相关，其中以神经性疾病和肿瘤最为突出。EMT即上皮细胞间质转化，是指上皮细胞失去上皮表型转化为间质细胞的复杂过程，EMT在肿瘤的侵袭转移中发挥重要功能。但是EMT相关的lncRNA还没有系统的报道。我们首先建立了TGFβ1诱导地EMT细胞模型，通过芯片筛选获得一系列EMT相关的lncRNA。按照一定的筛选原则对其中的23条序列进行Real-time PCR验证。本课题研究的主要内容是在进一步生物信息学分析的基础上，对lncRNA14具体功能和机制作进一步的阐释。并以前期数据为基础建立EMT相关的lncRNA调控网络，寻找EMT过程中关键的非编码序列，为肿瘤的诊断和治疗提供新的靶点。

肿瘤是威胁人类健康的重大疾病，其发病率和死亡率高居全球各种疾病首位，而侵袭和转移是恶性肿瘤致死的重要诱因。研究表明，上皮间质细胞转化（EMT）在多种癌症的原发性浸润和继发性转移中起到至关重要作用。肿瘤细胞通过EMT增强运动和迁移能力，促进肿瘤进入循环系统而发生转移。越来越多的研究表明，长链非编码RNA（LncRNA）在EMT过程中发挥了重要的调控作用。我们运用生物信息学和实验研究相结合的方法，对肿瘤恶化相关的公共数据、实验室自产的肿瘤芯片数据和SMAD3 ChIP-Seq数据进行重新分析和数据发掘，并对其中2条LncRNAs的功能和机制进行了深入研究。在第一部分工作中，我们发现，肝癌MSCs能够促进肝癌细胞的EMT过程和肝癌的发生发展。我们利用芯片数据分析，鉴定出一条在肝癌MSC中高表达的LncRNA，命名为LncRNA-MUF。敲降和过表达实验均证明LncRNA-MUF在肝癌进程中发挥促进作用。在机制上，我们发现LncRNA-MUF能够集合ANXA2蛋白，激活Wnt/β-catenin信号通路，促进EMT过程。另外，LncRNA-MUF能够发挥ceRNA作用吸附miR-34a，导致Snail上调，激活EMT进程。在第二部分工作中，我们利用SMAD3 ChIP-Seq数据进行重新分析，鉴定出19条SMAD3结合并且在癌和癌旁组织中差异表达的LncRNA，其中LINC01186在TGF-β处理的A549细胞中下调最明显。敲降LINC01186后，肿瘤细胞侵袭转移能力明显增强，同时我们也证实，LINC01186的表达受到SMAD3蛋白的调控。上述结果提示，LncRNAs在调控肿瘤细胞上皮间质细胞转化过程中发挥了重要的作用，有望成为转移恶性肿瘤的治疗靶标。