Function of the p53-induced lncRNA LINC01021 in suppression of colorectal cancer

最近的测序分析显示，人类基因组编码约32.000个长链非编码/lncRNA。因此，这类转录本在数量上与蛋白质编码mRNA相似，但在很大程度上代表了未知领域，有望在介导肿瘤抑制中发挥重要作用。lncRNA结构或调节的改变可能有助于人类肿瘤的发病机制。LncRNA具有超过200个核苷酸的长度，并且主要在细胞核中实现多种功能，在细胞核中它们结合并影响染色质相关蛋白以调节基因表达，但是LncRNA也与其他RNA（例如microRNA和DNA）相互作用。值得注意的是，由最常见的突变肿瘤抑制基因编码的p53转录因子也调节lncRNA。然而，p53调控的lncRNA的表征和鉴定还远未完成。 结直肠癌（CRC）在癌细胞中显示p53突变。60%的病例。为了全面研究p53在CRC中的功能，我们最近使用RNA-Seq、miR-Seq和定量蛋白质组学（pSILAC）分析确定了条件性p53等位基因在SW480 CRC细胞中激活后的差异RNA、microRNA和蛋白质表达。此外，我们通过ChIP-Seq分析确定了p53的整体DNA结合模式。通过这些分析的组合，我们将LINC01021鉴定为新的p53诱导的lincRNA，当异位表达时，其可重复地显示出p53的显著诱导（> 600倍）和抑制增殖。最近，我们报道了LINC01021的缺失增加了CRC细胞系对化疗药物的敏感性。此外，在CRC患者的CMS4亚组中，LINC01021的低表达与较差的存活率相关。尽管LINC01021具有肿瘤相关能力，但其作用的分子机制仍然知之甚少。我们的初步结果表明，LINC01021调节的一个子集的p53调控基因的表达。在这里，我们打算使用基因组和蛋白质组范围内，公正的方法来揭示LINC01021有助于调节p53网络的分子机制。LINC01021的蛋白质相互作用伴侣将通过RNA下拉和质谱分析来鉴定，以确定LINC0121影响基因表达的分子机制。LINC01021相互作用蛋白及其在LINC01021介导的基因调控中的功能作用将进一步详细描述。LINC01021介导的基因调控将通过ChIRP（通过RNA纯化的染色质分离）-Seq分析来鉴定。将确定LINC01021调节基因的功能相关性及其与p53介导的肿瘤抑制蛋白的相互作用。总之，这些分析旨在阐明LINC01021在p53网络中执行关键功能的机制。

Recent sequencing analyses revealed that the human genome encodes approximately 32.000 long-non-coding/lncRNAs. Therefore, this class of transcripts is similar in number as protein-coding mRNAs, but represents largely uncharted territory, which promises to have important functions in mediating tumor suppression. Alterations in lncRNA structure or regulation presumably contribute to the pathogenesis of human tumors. LncRNAs have a length of more than 200 nucleotides and fulfil diverse functions, mainly in the nucleus where they bind and affect chromatin-associated proteins to regulate gene expression, but LncRNAs also interact with other RNAs, e.g. microRNAs, and DNA. Notably, also the p53 transcription factor, which is encoded by the most commonly mutated tumor suppressor gene, regulates lncRNAs. However, the characterization and identification of p53-regulated lncRNAs is far from complete. Colorectal cancers (CRC) display p53 mutations in ca. 60% of all cases. In order to comprehensively study the function of p53 in CRC we recently determined the differential RNA, microRNA and protein expression after activation of a conditional p53 allele in SW480 CRC cells using RNA-Seq, miR-Seq and quantitative proteomics (pSILAC) analyses. In addition, we determined the global DNA binding pattern of p53 by a ChIP-Seq analysis. By the combination of these analyses, we identified LINC01021 as a novel p53-induced lincRNA, which reproducibly showed a dramatic induction by p53 (>600x) and suppressed proliferation, when expressed ectopically. More recently, we reported that deletion of LINC01021 increases sensitivity of CRC cell lines to chemotherapeutic drugs. Furthermore, in the CMS4 sub-set of CRC patients low expression of LINC01021 was associated with poor survival. Although, LINC01021 harbors tumor-relevant capacities the molecular mechanisms of its action are still poorly understood. Our preliminary results indicate that LINC01021 modulates the expression of a subset of p53-regulated genes. Here we intend to use genome- and proteome-wide, unbiased approaches to uncover the molecular mechanisms by which LINC01021 contributes to regulations within the p53 network. Protein interaction partners of LINC01021 will be identified by RNA pull-down and mass spectrometric analysis in order to determine the molecular mechanisms by which LINC0121 influences gene expression. The LINC01021 interacting proteins and their functional role in LINC01021-mediated gene regulation will be characterized in further detail. LINC01021-mediated gene regulations will be identified by ChIRP (chromatin isolation by RNA purification)-Seq analyses. The functional relevance of LINC01021-regulated genes and its interactions with proteins for p53-mediated tumor suppression will be determined. Taken together, these analyses aim to illuminate the mechanisms by which LINC01021 performs critical functions within the p53 network.