小开放阅读框编码多肽对神经母细胞瘤MYCN表达的调控作用及机制研究

Recent evidence shows that polypeptides encoded by small open reading frames within the non-coding regions of genome (sPEP) are able to impact the activity of transcription regulatory proteins through peptide-protein interaction, and subsequently regulate gene expression. Our previous studies have revealed the existence of evolutionarily conserved small open reading frame within the 3'-untranslated region of MYCN gene that is crucial for the progression of neuroblastoma (NB), which encodes one sPEP that might bind transcription factor heat shock factor 1 (HSF1) and inhibit the HSF1-facilitated MYCN expression. Meanwhile, HSF1 might facilitate the expression of MYCN through increasing the binding of CCCTC-binding factor (CTCF) to MYCN promoter. This project is proposed to further observe the regulatory effects of HSF1-mediated recruitment of histone acetyltransferase and chromatin remodeling on CTCF binding and MYCN expression, and elucidate the interaction domains between sPEP and HSF1 in NB. The impacts of sPEP on the binding of HSF1 to MYCN promoter and MYCN gene transcription will also be explored in NB. In addition, the proliferation, differentiation, invasion, and metastasis of NB cells will be studied through stable over-expression or inhibition of sPEP and MYCN gene rescue experiments. This study will elucidate the crucial roles and underlying mechanisms of endogenous small polypeptides in regulating the MYCN expression, and will provide a novel approach for the therapeutics of NB.

新近研究显示：基因组“非编码”区域小开放阅读框生成的多肽(sPEP)能以多肽-蛋白质相互作用方式，影响转录调节蛋白活性，调控基因表达。我们前期发现：在神经母细胞瘤(NB)恶性进展相关基因MYCN的3'-端非翻译区，存在进化保守的小开放阅读框，其编码的sPEP能结合并阻遏转录因子热休克因子1(HSF1)对MYCN表达的促进作用；而HSF1可能通过促进CCCTC结合因子(CTCF)在MYCN启动子的募集，上调MYCN表达。本项目拟进一步观测HSF1招募组蛋白乙酰化酶导致的染色质重塑对CTCF募集及MYCN表达的调控作用，解析sPEP与HSF1相互作用的结构域，明确sPEP对HSF1结合至MYCN启动子、MYCN基因转录的影响，观测sPEP稳定过表达/抑制、MYCN表达回补体系对NB细胞增殖、分化、侵袭转移的影响，旨在从新型内源性小分子多肽角度阐明MYCN表达调控机理，为NB的治疗提供新途径。

神经母细胞瘤(neuroblastoma)是婴幼儿及儿童期最常见的实体肿瘤之一，起源于原始神经嵴，大多数患儿早期诊断困难，肿瘤恶性程度高、发展迅速，且易发生骨髓、骨、肺及其他器官转移，死亡率仍高达儿童肿瘤的15%。原癌基因MYCN是Myc转录因子家族成员，在神经母细胞瘤增殖、分化、侵袭转移过程中发挥重要调控作用，但其高表达的分子基础及干预策略尚未阐明。本研究发现：在神经母细胞瘤组织和细胞中，转录因子热休克因子1（HSF1）高表达，且与MYCN表达水平呈正相关。染色质免疫沉淀检测证实：HSF1能通过促进CCCTC结合因子(CTCF)在MYCN启动子的募集，荧光素酶-报告基因、核连缀转录实验显示HSF1能促进MYCN启动子活性和基因表达；MYCN 3'-端非翻译区小开放阅读框编码生成多肽（sPEP），在神经母细胞瘤组织和细胞中表达下调，而与MYCN表达水平负相关；免疫共沉淀、染色质免疫沉淀检测发现MYCN sPEP能结合并拮抗过表达HSF1对MYCN启动子活性的促进作用，在转录水平抑制神经母细胞瘤细胞中MYCN基因表达。建立了MYCN sPEP稳定过表达或敲低的神经母细胞瘤亚克隆细胞株，进一步发现MYCN sPEP能抑制神经母细胞瘤细胞的体内外增殖、侵袭及转移活性，阻遏神经母细胞瘤的恶性进展。因此，本项目率先在国内外揭示了MYCN sPEP对神经母细胞瘤MYCN表达的调控作用及机制，丰富了MYCN基因表达调控机理，为神经母细胞瘤的治疗提供了新途径和研究基础。