LncRNA GATA2-AS1抑制FUBP3活性调控神经母细胞瘤GATA2表达及 有氧糖酵解的机制研究

Neuroblastoma (NB) is the most common extracranial solid malignancy in childhood. Aerobic glycolysis, also known as Warburg effect, promotes malignant progression of NB, but its regulatory mechanisms have not been elucidated. Our previous studies indicated that GATA binding protein 2 (GATA2) was an important transcription factor that negatively regulated the aerobic glycolysis. Long non-coding RNA GATA2-AS1 was able to bind to far upstream element binding protein 3 (FUBP3) and up-regulate GATA2 expression levels, while FUBP3 might be recruited to 3'-untranslated region of GATA2 to reduce its mRNA stability. Therefore, we hypothesized that GATA2-AS1 might promote GATA2 expression by regulating FUBP3 activity and inhibit the aerobic glycolysis of NB. This project intends to further observe the roles of GATA2 in regulating expression of target genes hexokinase 2 (HK2) and glucose-6-phosphate isomerase (GPI) in NB. The interaction between GATA2-AS1 and FUBP3 protein will be investigated by RNA binding protein immunoprecipitation and RNA EMSA, and their effects on GATA2 expression will be also revealed. The regulatory roles of GATA2-AS1 in aerobic glycolysis, proliferation, invasion and metastasis of NB will be elucidated. This is expected to provide novel approaches for the treatment of NB.

神经母细胞瘤(NB)是最常见的儿童颅外恶性实体肿瘤；有氧糖酵解(Warburg效应)促进NB恶性进展，但其调控机制尚未阐明。我们前期发现：GATA结合蛋白2(GATA2)是负性调控NB有氧糖酵解的转录因子；长链非编码RNA GATA2-AS1能结合远端上游元件结合蛋白3(FUBP3)，上调GATA2表达水平；而FUBP3可能募集至GATA2 3'-非翻译区，降低其mRNA稳定性。为此我们提出“GATA2-AS1通过调控FUBP3活性促进GATA2表达，抑制NB有氧糖酵解”的假说。本项目拟进一步探讨NB中GATA2对靶基因己糖激酶2(HK2)、磷酸己糖异构酶(GPI)表达的调控作用；运用RNA结合蛋白免疫沉淀、RNA EMSA等方法，探讨GATA2-AS1与FUBP3相互作用及其对GATA2表达的影响；明确GATA2-AS1对NB有氧糖酵解及增殖、侵袭转移的调控作用，为NB的治疗提供新思路。

神经母细胞瘤(NB)是最常见的儿童颅外恶性实体肿瘤；有氧糖酵解(Warburg效应)促进NB恶性进展，但其调控机制尚未阐明。综合多种数据库及测序平台的分析结果，我们发现与神经发育相关的转录因子GATA2可能为调控神经母细胞瘤糖酵解代谢的关键转录因子。转录因子GATA2可以通过结合于糖酵解相关酶HK2、ENO3和GPI基因启动子上来调控神经母细胞瘤糖酵解代谢水平。GATA2能调控肿瘤的增殖、侵袭和转移活性，在体外抑制神经母细胞瘤细胞的生物学活性。天然反义long non-coding RNA GATA2-AS1能调控GATA2转录活性及蛋白表达水平，GATA2-AS1能调控肿瘤的增殖、侵袭和转移活性，在体外和体内抑制神经母细胞瘤细胞的生物学活性。通过RNA pull-down实验、质谱、蛋白互作分析数据库BioGRID和RNA与蛋白结合能力分析平台catRAPID分析可得长链非编码RNA GATA2-AS1可与FUBP3结合，并可募集SUZ12蛋白。因此得出结论，在神经母细胞瘤中，长链非编码RNA GATA2-AS1对GATA2的调控作用是由RNA结合蛋白FUBP3和SUZ12介导的。通过前期实验结果和catRAPID graphic 网站预测设计抑制GATA2-AS1与FUBP3结合的多肽FIP-13，运用已筛选稳定过表达/敲低GATA2-AS1的融合有mCherry红色荧光蛋白标签的和稳定过表达/敲低FUBP3的神经母细胞瘤亚克隆细胞株，或者给予FIP-13干预，通过体内和体外实验，评估GATA2-AS1对神经母细胞瘤体内增殖、侵袭及转移的影响。最终阐明 GATA2-AS1/FUBP3/GATA2轴抑制神经母细胞瘤有氧糖酵解水平的机制，从内源性反义 lncRNA 的角度，丰富 GATA2 基因的表达调控机理，为神经母细胞瘤的临床治疗提供新靶点。