长链非编码RNA（Long non-coding RNAs, LncRNAs）是一大类长度超过200 nt的非编码RNA，它们利用自身RNA分子参与分子间相互作用而发挥功能。这种笼统的分类方法使lncRNAs在大小、结构、功能上存在多样性和复杂性，LncRNAs主要的生物学功能涵盖了顺式/反式调控基因转录、DNA修饰、RNA剪接、蛋白质支架、蛋白质修饰、miRNA海绵等。LncRNAs广泛地与DNA、RNA和蛋白质发生着复杂多样的相互作用。胚胎发育、心血管疾病、肿瘤发生发展过程中发现不少具有重要角色的lncRNAs。目前对lncRNAs的了解仅仅是冰上一隅，鉴于此我们开展了针对lncRNAs-蛋白质互作的研究，旨在探究lncRNAs-蛋白质相互作用在肿瘤细胞中的生物学功能。
      Nlp（Ninein like protein）是中心体γ-Tubulin环状复合物的重要成员，Nlp结合到γ-Tubulin定位到中心体上，参与了中心体复制、中心体成熟、微管锚定、有丝分裂纺锤体形成等众多细胞周期事件并发挥重要作用，Nlp表达的异常通常会引起中心体结构和数量的异常，导致单极或多极有丝分裂和核分裂细胞质不分裂等异常有丝分裂现象，进而造成基因组紊乱，导致细胞恶性转化。Nlp在细胞周期进程中与PLK1、NEK2、Aurora A/B、Cyclin B1等细胞周期调控蛋白时序性结合保证了细胞正常复制和生长。考虑到中心体在细胞中重要作用，及对Nlp这个中心体平台蛋白是否能搭载lncRNAs这类功能多样而复杂的分子，发挥对细胞周期调控的可能性，我们进行了本课题研究工作，旨在阐释lncRNAs与中心体蛋白Nlp相互作用对细胞有丝分裂的调控作用。
       我们通过Nlp RIP-seq在转录组范围内鉴定Nlp蛋白结合的RNAs，发现了上千条转录本，其中被注释为lncRNAs也达数百条之多，我们挑选了34条进行qRT-PCR验证，其中显著地被Nlp富集的就有24条，我们对其中7条富集倍数高、多次鉴定结果一致的lncRNAs进行亚细胞定位和多种肿瘤细胞中表达水平检测，最终选择了CCAT1和linc00665两个Nlp结合的lncRNAs，我们进一步通过RNA-FISH联合蛋白质免疫荧光观察lncRNAs与Nlp、γ-Tubulin是否存在共定位。发现CCAT1和linc00665分别都能与Nlp和γ-Tubulin共定位。CCAT1体外RNA pulldown有力地支持了CCAT1与Nlp和γ-Tubulin结合现象，RNA反义核酸纯化联合质谱鉴定证明CCAT1与Nlp是直接结合的，同时还能结合微管蛋白α-Tubulin和β-Tubulin但没有γ-Tubulin，证明CCAT1与γ-Tubulin的结合是间接的。另一方面，对于Nlp RIP发现的lncRNA linc00665，我们对其进行5’和3’末端RACE鉴定，发现在HeLa细胞中与Nlp结合的linc00665是一个新的转录本，它的第1、2号外显子与数据库收录的其他转录本一致。对CCAT1和linc00665的细胞学功能研究发现它们在HeLa细胞中都发挥促进细胞生长、迁移和侵袭的角色，CCAT1的过表达能帮助细胞抵抗紫杉醇对细胞的杀伤作用，另一方面导致HeLa细胞多极有丝分裂、中心体扩增细胞比例增加。
        综上，本课题研究工作成功鉴定出了两个中心体蛋白Nlp结合的lncRNAs CCAT1和linc00665，它们在细胞内与中心体蛋白Nlp、γ-Tubulin相互作用，都存在一定程度的中心体定位，其中外源CCAT1过表达导致中心体过度复制、有丝分裂异常细胞比例增多现象，我们认为CCAT1和linc00665可能在一定程度上通过扰乱中心体结构和数量，破坏中心体稳态，增加有丝分裂异常，进而导致染色体不稳定、基因组不稳定，最终导致细胞生长加快、侵袭和迁移能力增强等恶性转化现象。

   lncrnas (long non-coding rnas) are a broad definition and categories of ncrna exceed 200 nucleotides in length, and function by the interaction with other molecules. lncrnas could regulate gene tranion via cis/trans interacting on promoters. they also participate in dna modification, rna splicing and modification in cell nucleus. some cytoplasma lncrnas act as rna-binding proteins (rbps) and micrornas decoys then sequester those rbps and micrornas. lncrnas also form special structure for protein-protein interaction. depending on their widely interaction with dna and proteins, lncrnas take important role in cell biology behavior and some disease including tumor biogenesis and progress. the large unknown lncrna-protein interactions appeal us to exploration.
  nlp (ninein like protein) is a member of γ-tubulin complexes binding proteins. it functions on microtube nucleation, centriole biogenesis, centrosome maturation and spindle formation, taking a key role in cell mitosis events. nlp interacts with γ-tubulin and locates on centrosome. studies showed that nlp could be regulated by cell cycle related kinase plk1, nek2, aurota a/b, cyclin b1 and so on. additionally, nlp is a docking protein on centrosome and microtubes. it is unknown whether this docking protein assembling with lncrnas besides proteins. considering nlp formed a platform in cell, it is possible that lncrnas bind to nlp and help to coordinate its function on centrosome and microtubes. as a possible result, lncrnas regulate cell cycle interacted with nlp.
firstly, we performed nlp rip-seq and found thousands of nlp related rnas, among which were hundreds of definited annotation lncrnas . then we selected and validated 34 of those lncrnas by rt-qpcr. as a result, 24 candidate lncrnas were significantly enriched by nlp and coordinated with sequencing data. secondly, we picked out 7 candidate lncrnas according to their repeatability in more than 3 times independently validation and their high enrichment by nlp. ccat1 and linc00665 were chosen for their dominant cytoplasma location. knockdown ccat1 and linc00665 showed cell growth inhibition and decreased the ability of migration and invasion, respectively. in addition, ccat1 overexpression exhibited cell growth promotion, enhanced migration and invasion ability and cell resistantance for paclitaxel induced cell apoptosis. thirdly, we performed rna-fish combined immunofluorescence to observe the localization of ccat1, linc00665, nlp and γ-tubulin. we found both ccat1 and linc00665 can co-localized with both nlp and γ-tubulin on centrosome, indicating that they are both centrosomale lncrnas. fourthly, we performed rna pull-down assay by ccat1 full length rna and fragments in vitro. and we found ccat1 full length rna can pull down both nlp and γ-tubulin. the fragment from 5’ prime to 3/5 portion of ccat1 full length shown highest nlp and γ-tubulin binding ability than other fragments. we also performed rna antisense purification coupled with mass spectrometry (rap-ms) to validate the direct/indirect interaction between ccat1 and nlp/γ-tubulin in vivo. on the other hand, rap-ms can help us to identify proteome-wide ccat1 binding proteins. rap-ms results showed that ccat1 interacts with nlp directly but with γ-tubulin indirectly. interestingly, we also found α-tubulin, β-tubulin, vimentin are ccat1 binding proteins. lastly, considering that for several trans were recorded by ncbi and ensembl database, we performed 5’and 3’ end race to validate the sequence of linc00665. noteworthy, we identified a new linc00665 tran only shared 500 nt of its 5’ end with known linc00665 trans. this new linc00665 tran is a centrosomale lncrna.
  in conclusion, we identified hundreds of nlp associated lncrnas. among them, ccat1 and linc00665 were validated as centrosome lncrnas. ccat1 located on centrosome by interacting with nlp directly, but with γ-tubulin indirectly. addtionally, we identified a new linc00665 tran by 5’ and 3’ race and also found this new linc00665 co-localized with nlp and γ-tubulin on centrosome. both ccat1 and linc00665 promote tumor progression by enhancing the growth, migration and invasion abilities of tumor cells.