RRS1在神经母细胞瘤发展中的作用及其分子机制研究

Neuroblastoma(NB) is the most common extracranial solid tumor of childhood, which characters difficult diagnosis, rapid progression and poor prognosis resulting in survival rates of less than 50%. The precise mechanism of development and progression of NB is still unclear. RRS1 (ribosome biogenesis regulator homolog) homolog has been discovered in the nucleoli protostome of HeLa cells in last decade. Although yeast Rrs1 has been widely studied in S. cerevisiae, the role of RRS1 in the progression of neuroblastoma has not been discovered yet. Moreover, the function of RRS1 homolog is not well understood. Relying on our initial results, it has demonstrated that lentiviral knock-down of RRS1 in SH-SY5Y inhibits cell proliferation and promotes apoptosis. Besides, our study show that RRS1 involving in neuronal differentiation by using microarray analysis by IPA. While Rrs1 is involved in yeast ribosome assembly, the mechanism of RRS1 in regulation of neuroblastoma progression and its role in control of ribosomal assembly remains to be resolved, hence the study will demonstrate the importance of RRS1 involving in neuroblastoma progression. It has been shown that yeast Rrs1 participates in 60S ribosome assembling by recruiting rpL5 and rpL11 with 5S rRNA to regulate the proliferation of yeast. Therefore, it seems reasonable that RRS1 homolog implies in neuroblastoma progression via regulation of ribosome assembling. To investigate the hypothesis above, human neuroblastoma cell lines and nude mice will be used for the study the role of RRS1 in neuroblastoma proliferation and differentiation. Realtime-PCR and IHF will be performed to explore the relationship between expression level of RRS1 and tumor status, which also includes sex, age, INSS stages and Shimada classification. Furthermore, affinity chromatography combined LC-MS/MS, GST Pull-Down, Co-IP, Northern blotting and RNA EMSA will be applied to investigate the molecular mechanism for regulation of RRS1 in human ribosome biogenesis. In summary, the results from this study will provide evidence to discover a novel biomarker for prognosis of neuroblastoma.

神经母细胞瘤是最常见的儿童颅外实体恶性肿瘤，迄今为止其发生发展分子机制不清。本课题在文献报道以及前期工作的基础之上，提出RRS1在NB细胞中表达增高，并通过参与调节NB细胞pre-rRNA转录、加工，影响60S核糖体亚基的装配，从而调节NB细胞周期，导致神经母细胞瘤增殖加快并抑制分化，最终致使NB迅速进展分子机制的假说。本课题将以人神经母细胞瘤细胞系和儿童神经母细胞瘤临床样本为研究对象，建立裸鼠成瘤模型，验证RRS1可调节神经母细胞瘤进展的科学假说；同时采用蛋白质亲和层析联合质谱、Co-IP、GST Pull-Down、Northern blotting、RNA EMSA等手段揭示RRS1在神经母细胞瘤中影响核糖体装配的分子机制；为神经母细胞瘤临床诊疗提供线索和理论依据。

神经母细胞瘤（Neuroblastoma, NB）是最常见的儿童颅外实体恶性肿瘤。NB通常发生于肾上腺或者脊柱两侧交感神经节。NB患者病情进展快、生存期短。肿瘤的发生发展需要大量物质合成以维持其快速增殖。核糖体的生物合成与装配则是满足大量蛋白质合成的重要前提。人核糖体生成调节因子1（RRS1）于2002年在HeLa细胞核仁蛋白组中被发现，但具体功能和相关机制尚不清楚。在模式生物酵母中的研究发现，Rrs1参与核糖体60S大亚基的装配，人RRS1是否也通过参与核糖体60S大亚基装配从而影响NB进展也不清楚。.本项目研究发现RRS1可促进NB增殖、抑制分化：敲减RRS1可抑制NB细胞增殖、细胞周期进程和裸鼠成瘤能力，抑制NB细胞凋亡和分化；临床样本显示，RRS1高表达与NB非分化型和不良预后显著相关。.我们还发现，RRS1定位于核仁，参与人核糖体60S大亚基装配：免疫荧光和免疫组化实验说明RRS1定位于核仁；免疫沉淀联合质谱发现RRS1与核糖体60S大亚基装配蛋白相互作用；且敲减RRS1可诱发核仁应激。.此外，RRS1可能通过调控JAK/STAT通路调控NB细胞分化，是潜在治疗靶点：我们发现，在NB细胞系中敲减RRS1可激活JAK/STAT通路并诱发维甲酸（Retinoic Acid，RA）诱导的细胞凋亡，这与RA处理的NB细胞系基因表达谱相一致；本项目还发现敲减RRS1可以促进RA诱导的NB细胞系向神经细胞分化，说明敲减RRS1可能成为RA的辅助治疗方式，维持NB后期治疗。本课题为进一步建立NB临床预后指标提供理论基础；为研发NB药物、治疗NB提供参考。