启动子相关长链非编码RNA对神经母细胞瘤中FOXD3表达的调控作用及机制研究

Our recent evdience has shown that forkhead box D3 (FOXD3), a novel tumor suppressor, abolishes the proliferation, invasion, metastasis, and angiogenesis of neuroblastoma (NB) cells. However, the mechanisms underlying the down-regulation of FOXD3 in NB still remain largely unknown. Our preliminary data have revealed the existence of endogenous FOXD3 promoter-associated long non-coding RNA (pasFOXD3) in NB tissues and cells, which might bind the transciption factor Yin Yang 1 (YY1) through RNA-protein interaction and faciliate YY1-mediated repression on FOXD3 expression. Based on these findings, this project is proposed to elucidate the regulatory effects of YY1 on the promoter activities, histone deacetylation, and expression of FOXD3 in NB tissues and cell lines. To unreveal the mechanisms underlying the regulatory effects of pasFOXD3 on FOXD3 expression, the interaction between pasFOXD3 and YY1 and its influence on the binding of YY1 to target loci of FOXD3 and FOXD3 transciption will also be explored in NB tissues and cells. After stable over-expression or knockdown of pasFOXD3, the proloferation, invasion, metastasis, and angiogenesis of NB cells will be studied in vitro and in vivo. This study will define the roles and mechanisms of promoter-associated long non-coding RNA in regulating the FOXD3 expression, and will provide a novel strategy and theoretical basis for the therapeutics of NB.

我们前期发现：叉头框D3(FOXD3) 是一种新的抑癌基因，能阻遏神经母细胞瘤(NB)的增殖、侵袭转移及血管生成，但其表达下调机制不明；FOXD3启动子区域存在的内源性长链非编码RNA(pasFOXD3)可能通过RNA-蛋白质相互作用，结合转录因子阴阳蛋白1(YY1)，促进YY1对NB组织和细胞中FOXD3基因表达的抑制作用。本项目拟在此基础上，解析YY1对NB组织和细胞中FOXD3启动子活性、组蛋白去乙酰化、基因表达的调控作用；观测NB组织和细胞中pasFOXD3与转录因子YY1的相互作用及其对YY1锚定至FOXD3启动子、FOXD3基因转录的影响，探讨pasFOXD3调控FOXD3基因表达的分子机制；观测稳定过表达或抑制pasFOXD3对NB细胞体内外增殖、侵袭转移及血管生成的影响，旨在从启动子相关的长链非编码RNA角度阐明FOXD3表达调控机理，为NB的治疗提供新策略和理论依据。

神经母细胞瘤起源于原始神经嵴，是婴儿及儿童期最常见的实体肿瘤之一，恶性程度高，进展迅速，易发生多部位 (骨髓、骨及肺) 转移。叉头框D3 (FOXD3) 是一种新发现的抑癌基因，能阻遏神经母细胞瘤的增殖、侵袭转移，但其表达调控机制不明，靶向调控途径有待深入解析。本研究发现：转录因子阴阳蛋白1 (YY1)、CCCTC结合因子 (CTCF) 在神经母细胞瘤组织和细胞中高表达，与FOXD3表达水平负相关。染色质免疫沉淀、荧光素酶-报告基因、核连缀转录实验证实：YY1、CTCF能靶向结合至FOXD3启动子区域，抑制FOXD3基因的表达；神经母细胞瘤组织和细胞中存在FOXD3启动子相关长链非编码RNA (FOXD3-AS1)，与FOXD3表达水平正相关；RNA pull-down、RNA免疫沉淀、免疫共沉淀、染色质免疫沉淀检测发现FOXD3-AS1能结合1型多聚ADP核糖合成酶 (PARP1), 抑制PARP1与CTCF蛋白的相互作用，进而阻遏转录因子CTCF、YY1作用于FOXD3启动子的调控位点，上调神经母细胞瘤细胞中FOXD3基因转录和表达水平。通过建立稳定过表达或敲低FOXD3-AS1的神经母细胞瘤亚克隆细胞株，进一步发现FOXD3-AS1能抑制神经母细胞瘤细胞的体内外增殖、侵袭转移活性，在神经母细胞瘤恶性进展中发挥重要作用。因此，本项目率先在国内外揭示了FOXD3-AS1对神经母细胞瘤FOXD3表达的调控作用及机制，丰富了FOXD3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，具有广阔的应用前景和潜在的社会、经济效益。