一个功能未知的假基因编码多肽NCSP1抑制非小细胞肺癌恶性进展的功能和机制研究

With peptidomics and bioinformatics methods, we identified a novel small peptide，non-small cell lung cancer (NSCLC) associated small peptide 1 (NCSP1), which is encoded by a pseudogene. We detected high mass spectrometry (MS) signal of NCSP1 in non-tumor tissues, while no MS signal was detected in NSCLC tissues. NCSP1 could inhibit proliferation and invasion ability of NSCLC cells independent of the RNA transcript of TOB2P1. With pull down assay, we found that NCSP1 could bind with lysine-specific histone demethylase 1A (LSD1), and gene sets enrichment analysis showed that many tumor suppressive genes which could be epigenetically suppressed by LSD1 was positively enriched by NCSP1. Thus, we hypothesized that NCSP1 could inhibit the malignant progression of NSCLC by binding with LSD1 and block its epigenetic repression to downstream tumor suppressor genes. To validate this hypothesis, we plan to confirm that NCSP1 could inhibit the malignant progression of NSCLC by in vitro and in vivo experiments, as well as in clinical samples. Also, we aimed to clarify the molecular mechanism of NCSP1, by which NCSP1 interacts with LSD1 and block its epigenetic repression effects on downstream target genes, and find out whether NCSP1 could be a therapeutic target of NSCLC. There has been no report about NCSP1; therefore, our work has original innovation and may provide new insight into the treatment of drug development of NSCLC.

NCSP1是申请人前期借助多肽组学筛选获得、由假基因TOB2P1编码、功能未知的多肽。预实验发现：NCSP1在正常肺组织中质谱信号较高，但非小细胞肺癌（NSCLC）中无质谱信号；NCSP1可不依赖于TOB2P1转录本独立抑制NSCLC细胞增殖、迁移等恶性行为；pull down实验证实NCSP1可与促癌蛋白赖氨酸特异性去甲基化酶1（LSD1）结合；基因集富集分析显示，LSD1表观抑制的多种抑癌基因可被NCSP1正向富集。因此提出“NCSP1可结合LSD1并拮抗其对下游抑癌基因的表观抑制，进而抑制NSCLC恶性进展”这一假说。本研究拟从临床样本、细胞和动物模型层面验证NCSP1抑制NSCLC恶性进展的生物学功能；以拮抗LSD1表观抑制为机制线索，阐明NCSP1分子机制，论述其作为治疗靶标的可能性。目前尚无NCSP1相关报道，因此本研究具有源头创新性，同时也将为NSCLC药物研发提供新线索。

肺癌是严重威胁人类生命健康的恶性肿瘤，其中非小细胞肺癌（NSCLC）是.最常见的病理亚型。近来研究发现非编码RNA，尤其是长非编码RNA（lncRNA）.在肿瘤的恶性进展过程中发挥着重要作用。前期研究者通过质谱发现假基因.TOB2P1 可编码小分子多肽，并将其命名为NCSP1。本研究选取了96例早期肺癌组织标本（48例CT表现为磨玻璃/部分实性结节，48例CT表现为实性结节）与48例瘤旁组织，提取组织的总蛋白之后使用Q-exacitve质谱检测。检索质谱数据未发现NCSP1对应的独特肽段，其原因可能为NCSP1表达丰度过低、提取及质谱检测方法差异导致。在RNA层面上，NCSP1母本基因TOB2P1的表达水平在肺癌组织中高于正常肺组织，但是与临床病理资料无统计学相关性。本研究同时还进行了1）构建了可能由lncRNA编码的多肽文库，通过与质谱数据检索，系统性鉴定早期肺癌中可能由lncRNA编码的多肽；其中有343个多肽在至少1/2样本中有表达，且有110条多肽在肺癌中表达水平显著升高。2）研究了早期肺癌的蛋白质组学特征；发现有1795个蛋白在肺癌与瘤旁组织中存在差异表达；与瘤旁组织相比，实性肺癌中高表达蛋白显著集中在氨基酸代谢及中性粒细胞相关通路；与瘤旁组织相比，磨玻璃肺癌中差异表达的蛋白也显著富集在代谢与免息相关通路中。相对于瘤旁组织和GGO型肺癌，实性肺癌中有19个蛋白表达显著升高：MUC5A, LEG4, MUC5B, TSP2, AGR2, DESP, RCN3, NQO1, AK1BA, OXLA, CSPG2, ERO1A, GMDS, GLNA, P4HA1, MXRA5, DPYD, ADSV, QSOV1。