环状RNA结合MAZ蛋白调控神经母细胞瘤中hnRNPK基因转录的作用及机制研究

Our recent studies reveal that heterogeneous nuclear ribonucleoprotein K (hnRNPK) facilitates the progression of neuroblastoma (NB), while the mechanisms regulating its expression remain largely unknown. In addition, circular RNA derived from the intron of hnRNPK (ci-hnRNPK) is able to bind to MYC associated zinc finger protein (MAZ) to promote the MAZ-facilitated hnRNPK gene transcription in NB tissues and cell lines. Based on these findings, this project is proposed to further elucidate the interaction between MAZ and RB binding protein 4 (RBBP4) and their roles in recruiting histone acetyltransferases p300/CBP and epigenetically regulating the promoter activity and expression of hnRNPK in NB. The interaction and domains of ci-hnRNPK and MAZ will be elucidated, and the regulatory effects of ci-hnRNPK on the activity of transcription factor MAZ and expression of hnRNPK gene will be investigated. The effects of over-expression or knockdown of ci-hnRNPK and rescue of hnRNPK expression or MAZ activity on the proliferation, invasion, and metastasis of NB will be observed in cultured cell lines and animal models. This study will elucidate the crucial functions and underlying mechanisms of endogenous circular RNA in regulating the hnRNPK expression, and will provide novel approach and theoretic basis for the therapeutics of NB.

本课题组新近发现：核不均一核糖核蛋白K(hnRNPK)促进神经母细胞瘤(NB)恶性进展，但其表达调控的分子机制不明；hnRNPK内含子来源的环状RNA(ci-hnRNPK)能通过结合MYC相关锌指蛋白(MAZ)，增强MAZ对NB组织和细胞中hnRNPK基因转录的促进作用。在此基础上，本项目拟深入解析转录因子MAZ与视网膜母细胞瘤结合蛋白4(RBBP4)相互作用、进而招募组蛋白乙酰化酶p300/CBP对NB中hnRNPK启动子活性及基因表达的调控作用；明确ci-hnRNPK与MAZ蛋白的相互作用及结构域，观测ci-hnRNPK对MAZ转录因子活性及hnRNPK基因表达的影响；在细胞和动物水平，观测过表达/敲低ci-hnRNPK、回补hnRNPK表达或MAZ功能对NB增殖、侵袭和转移的调控作用，有望阐明新型内源性环状RNA对hnRNPK表达的调控作用及机制，为NB的治疗提供新途径及理论依据。

神经母细胞瘤(neuroblastoma)起源于原始神经嵴细胞，是小儿最常见的恶性实体肿瘤，80%病例发生于5岁以下儿童，可发生于肾上腺髓质及存在交感神经系统的任何部位(如腹膜后、后纵隔、颈部、盆腔)，大多数肿瘤位置隐蔽，恶性程度高且进展迅速，高危组5年生存率不足30%。核不均一核糖核蛋白K(hnRNPK)是hnRNP蛋白家族成员，在染色体重塑、基因转录、mRNA剪接及稳定性等方面发挥重要的调控作用，与肿瘤侵袭转移及不良预后密切相关。然而，hnRNPK在神经母细胞瘤组织和细胞中高表达的分子机制尚未阐明。本研究发现：转录因子MYC相关锌指蛋白(MAZ)在神经母细胞瘤组织和细胞中高表达，能与视网膜母细胞瘤结合蛋白4(RBBP4)相互作用。采用染色质免疫沉淀、荧光素酶-报告基因转染等方法，进一步证实MAZ能招募p300/CBP及乙酰化组蛋白(H3K9ac、H3K27ac)至hnRNPK启动子，促进hnRNPK启动子活化及基因转录；在神经母细胞瘤的组织标本和细胞株中，hnRNPK内含子来源的环状RNA(ci-hnRNPK)表达升高，能结合并增强转录因子MAZ对hnRNPK表达的上调作用；通过稳定过表达ci-hnRNPK，建立了神经母细胞瘤亚克隆细胞株模型，细胞培养和裸鼠动物实验表明：ci-hnRNPK能促进神经母细胞瘤细胞的增殖、侵袭及转移活性，驱动神经母细胞瘤的恶性进展。因此，本项目揭示了新型内源性环状RNA对hnRNPK表达的调控作用及分子机制，丰富了hnRNPK基因转录调控机理，为神经母细胞瘤的防治提供了新靶标和潜在途径。