长链非编码RNA-HOXB-AS3通过编码一个多肽调控PKM基因剪切抑制结肠癌发生发展的功能机制研究

Recent advances in deep sequencing technologies have led to the identification of a large number of previously unknown transcripts. The vast majority (>99%) of these transcripts are considered long non-coding RNAs (lncRNAs), which are not translated into proteins given the lack of obvious long protein-coding open reading frames (ORFs) and clear homologs in other organisms. Accumulating evidence has shown that altering lncRNA expression levels leads to the dysregulation of downstream effectors and that lncRNAs may be involved in the regulation of various hallmarks of cancer..Our previous study found that a lncRNA HOXB-AS3 may be associated with colon tumorigenesis and metastasis. However, the functional mechanisms of lncRNA HOXB-AS3 on tumorigenesis and metastasis are unknown. Our preliminarily data demonstrated that HOXB-AS3 RNA level was decreased in high-metastatic cancer cell sub-lines and colon tissues compare with those in their parental cell lines and corresponding non-tumoral colon tissues, respectively, suggesting that HOXB-AS3 may be a novel tumor suppressor. Interestingly and surprisedly, we found for the first time that a short 159-nucleotide small open reading frame (sORF) with the potential to encode a highly conserved 53-amino acid peptide in lncRNA HOXB-AS3 through bioinformatics and biochemistry assays. HOXB-AS3 peptide, not HOXB-AS3 lncRNA, inhibits colon cancer cell growth, colony formation, migration and invasion in vitro. To investigate the possible mechanism of HOXB-AS3 peptide, a known PKM pre-mRNA splicing regulator hnRNP A1 was preliminarily found to be possibly interacted with HOXB-AS3 peptide. Therefore, a scientific hypothesis in which a putative lncRNA HOXB-AS3 encodes a peptide that binds to hnRNP A1 and antagonizes the hnRNP A1-mediated regulation of pyruvate kinase M (PKM) splicing by blocking the binding of hnRNP A1 to the sequences flanking PKM exon 9, ensuring the formation of lower amounts of PKM2 and suppressing glucose metabolism reprogramming and subsequent colon cancer progression and metastasis..In the project, a peptide encoded by lncRNA HOXB-AS3 will be further validated in vitro and in vivo. The functional roles of HOXB-AS3 peptide and lncRNA on colon cancer progression and metastasis will be fully investigated in vitro and in vivo. The mechanism of HOXB-AS3 peptide on hnRNP A1-dependent PKM splicing and glucose metabolism reprogramming will be elucidated. The correlations between HOXB-AS3 peptide level and clinico-pathological features will be investigated, and the correlation of HOXB-AS3 peptide levels with a poor prognosis in patients with colon cancer will be analyzed..In conclusion, the functions and mechanisms of HOXB-AS3 in colon cancer progression and metastasis will be fully elucidated in the project. We indicate for the first time that an lncRNA exerts it's effects by encoding a peptide in cancer progression and metastasis. The study will be helpful for us to discover novel progonsis biomarkers and drug targets for intervening colon cancer progression and metastasis, and broaden and enrich our view about cancer progression and metastasis.

lncRNAs异常表达与肿瘤发生发展密切相关。我们前期筛选发现一个新lncRNA HOXB-AS3可能与结肠癌相关，但其功能机制未知。我们前期预实验，证实HOXB-AS3在高转移潜能肿瘤细胞和结肠癌组织中下调；惊喜地发现HOXB-AS3可能编码一个53个氨基酸的多肽；HOXB-AS3多肽，而不是其lncRNA，具有抑制结肠癌的功能；进一步筛选发现HOXB-AS3多肽可能与PKM基因剪切调控分子hnRNP A1相互作用。据此我们提出科学假说：lncRNA HOXB-AS3通过编码一个多肽，与hnRNP A1蛋白相互作用，调控了hnRNP A1对PKM基因的剪切和PKM2剪切体的形成，从而抑制细胞有氧糖酵解，进而抑制结肠癌的发生发展。本项目将在临床样品、细胞模型和动物实验中，回答此科学问题。首次在肿瘤中阐明lncRNA通过编码一个多肽发挥功能，为发现结肠癌预后标记和药物干预靶标提供理论依据。

在真核生物中，大部分的编码RNA是长链非编码RNA （lncRNA），大量研究研究证实，lncRNA以RNA分子的方式调控了肿瘤的各种功能。在本项目研究中，我们发现lncRNA HOXB-AS3编码产生一个53个氨基酸的、在灵长类生物中保守的新多肽。HOXB-AS3多肽，而不是HOXB-AS3 lncRNA本身抑制了结肠癌的发生发展。机制上，HOXB-AS3多肽竞争性的结合剪切调控分子hnRNP A1蛋白RGG基序上的精氨酸残基，阻碍了此精氨酸残基与代谢关键限速酶基因PKM pre-mRNA 9号外显子侧序的结合，调控了PKM基因mRNA的剪切，导致PKM基因9号外显子的包含、而不利用10号外显子，从而介导了PKM1剪切体的产生，而抑制了PKM2剪切体的产生，进而抑制了结肠癌细胞的糖代谢重编程，所以抑制了结肠癌的发生发展。具有低HOXB-AS3多肽水平的结肠癌患者具有更差的恶性表型和差的预后。我们的研究显示，HOXB-AS3多肽的缺失是结肠癌细胞代谢重编程中的一个关键重要事件，揭示了一个lncRNA编码的多肽精密调控肿瘤代谢重编程的复杂调节网络。发现lncRNA通过编码功能性多肽调控肿瘤发生发展的新机制，揭示lncRNA编码的多肽可能是一个新的肿瘤标志物和抗癌药物靶标。