PHF20调控神经母细胞瘤细胞干性维持的表观遗传学机制研究

The degree of neuroblastoma (NB) differentiation correlates with tumor progression and clinical outcomes, but its regulation mechanism remains unclear. Through high-throughput screening, we identified epigenetic regulation factor PHF20 as an important driver of stem-like phenotype in NB. PHF20 is up-regulated in NB specimens and closely correlated with clinical prognosis. Then, what is the role of PHF20 during the progression of NB? What are the molecular mechanisms that drive the maintenance of stemness and differentiation inside the NB tumor mass? Using cell and nude mice model, we proved that knockout of PHF20 significantly repressed the aggressiveness and stemness of neuroblastoma cells. Further studies confirmed that PHF20 was strongly bound to Sox2 and Oct4 promoters and affected their expressions. On the basis of these findings, we hypothesized that PHF20 regulates the H3K4me3 levels of stemness-related genes through interaction with epigenetic enzymes to sustain the stem cell-like phenotype and high aggressiveness of the tumor. This project intends to use methods such as CRISPR-Cas9, co-IP and ChIP-seq to study the crucial role of PHF20 in the maintenance of stem cell-like state of NB cells and illuminate the molecular mechanism, which will provide novel insights for the diagnosis and targeted therapy of NB.

神经母细胞瘤细胞分化水平决定肿瘤发展进程和临床预后，但其调控机制尚不明确。前期实验证实，表观遗传因子PHF20是神经母细胞瘤细胞维持未分化状态重要的驱动因子；PHF20在神经母细胞瘤中高表达且与临床预后相关。那么，PHF20基因表达在肿瘤发展进程中的作用是什么？其调控机制又是什么？通过细胞和裸鼠模型发现，PHF20基因敲除显著抑制神经母细胞瘤恶性程度和细胞干性；进一步研究发现，PHF20与Oct4，Sox2等细胞干性基因启动子区域相结合并影响其表达。据此我们提出假设：PHF20通过与表观遗传酶类相互作用调节干性基因启动子区域H3K4me3修饰水平使癌细胞维持低分化水平和高恶性程度。本研究拟采用CRISPR-Cas9技术、免疫共沉淀、ChIP-seq等方法，阐明PHF20在神经母细胞瘤细胞干性维持中的重要作用及调控机制，为神经母细胞瘤的临床诊断和靶向治疗提供新的科学依据。

本课题紧紧围绕PHF20敲除或过表达的神经母细胞瘤细胞与野生型细胞在神经母细胞瘤发生发展的几个关键环节上的差异设计实验方案开展研究。目前，我们已经成功获得了PHF20参与神经母细胞瘤细胞干性维持的可靠证据，阐明了PHF20表观调控神经母细胞瘤细胞干性维持的分子机制，明确了在PHF20介导的神经母细胞瘤细胞干性维持中起决定作用的细胞干性基因。成果以第一作者和共同第一作者发表在Nature Cell Biology和 Cancer Research等杂志。相关的成果也申请了专利1项 （申请号：201810309051.0），为后续科研转化工作的开展打下了坚实的基础。.本项目的意义在于（1）PHF20在干细胞自我更新和体细胞重编程过程中发挥了重要作用，但作为一个表观遗传调控因子，其在癌症中的作用，尤其在癌细胞干性维持中发挥的作用尚不明确。本研究系统的探讨了PHF20敲除或过表达对神经母细胞瘤细胞生物学活性和干性维持的影响，从而明确了PHF20参与神经母细胞瘤细胞干性维持的重要作用；（2）首次从分子层面探讨了PHF20介导的癌细胞表观遗传调控机理。虽然前人研究对神经母细胞瘤癌症干细胞的产生和机理有所报道，但从新兴的表观遗传因子PHF20的角度研究这一过程尚属首次。本研究不仅可以揭示PHF20表观遗传调控癌细胞干性维持的分子机制，还可以发现许多有价值的分子靶点，为神经母细胞瘤的预后和治疗提供新的策略。