eEF1A2介导蛋白翻译水平调控在骨肉瘤肿瘤干细胞成骨向分化中的作用及机制研究

Osteosarcoma is the most common malignant bone tumor in adolescent. Recently, tumor stem cells are proved to be the radical cause of metastasis and drug resistance. In our preparatory studies, up-regulation of eEF1A2 was found in the high metastatic, drug resis tant and spheroid enriched osteosarcoma cells by iTRAQ screen and validation. Then, we knocked down eEF1A2 in chemo-resistant osteosarcoma cells. It could induce the osteoblastic differentiation and reduced the tumorigenic capacity of the cells.eEF1A2 is an well-known regulator on the protein translation. Recently the role of eEF1A2 in the tumorigenesis and development was also been reported. However, it is still unclear about the role of eEF1A2 in trans-differentiation of osteosarcoma tumor stem cells to osteoblasts and the concerning mechanism of regulation on protein translational level. We propose to enrich osteosarcoma tumor stem cells by the CD117/Stro-1 screening; identify relationship between eEF1A2 and osteosarcoma tumor stem cell by function test; validate role of eEF1A2 on osteoblast differentiation of osteosarcoma tumor stem cells in vitro and in vivo; screen the eEF1A2 downstream target genes by RIP-Seq and RIP; analyze eEF1A2 and function of those target genes via combination of functional tests and clinical research. Together, this project aim to investigate the role of eEF1A2 in the transdifferentiation of osteosarcoma tumor stem cells to osteoblasts and mechanism of regulation on protein translational level, and explore new strategies and bio markers for osteosarcoma treatment.

骨肉瘤是青少年骨骼系统最常见恶性肿瘤。近来研究发现肿瘤干细胞(TSCs)是恶性肿瘤发生、进展的根源。我们前期通过iTRAQ筛选、验证，发现eEF1A2在高转移、耐药及成球富集细胞中显著高表达；而敲低eEF1A2，肿瘤细胞干性下调，且出现成骨向分化。eEF1A2是翻译水平重要调控因子，最新发现在肿瘤发病中举足轻重。目前eEF1A2在骨肉瘤TSCs成骨分化中作用及翻译水平调控机制尚未阐明。现拟在前期基础上利用CD117/Stro-1分选骨肉瘤TSCs；通过功能实验，明确eEF1A2与骨肉瘤TSCs关系；运用细胞、动物模型验证eEF1A2在骨肉瘤TSCs成骨分化中作用；应用RIP-Seq、RIP等筛选并验证靶基因；结合功能实验、临床病例，分析eEF1A2及其靶基因功能和临床意义。本项目旨在研究eEF1A2在骨肉瘤TSCs成骨向分化中的作用及翻译水平调控机制，探索骨肉瘤新的分子靶标和治疗策略。

骨肉瘤是青少年全身骨骼系统最常见的恶性肿瘤，对青少年甚至整个家庭危害严重。骨肉瘤的科研探索和临床治疗停滞在平台期，骨与软组织肿瘤专家通过提高化疗剂量和尝试不同方案的细胞毒药物，却不能有效地降低肺转移率和改善整体预后。其根本原因在于现阶段对骨肉瘤的生物学特性认识不足，直接导致目前的治疗措施未能彻底清除或控制肿瘤细胞。因此，对骨肉瘤的生物学特征进行更深层次的认知迫在眉睫，在此基础上探索新的治疗策略及相关分子靶标显得尤为重要，这将有助于打破目前临床疗效停滞的瓶颈，进一步提高骨肉瘤的整体预后。本项目为骨肉瘤的治疗提供新策略—以SREBF1为靶点的精准治疗。本课题通过iTRAQ技术对骨肉瘤细胞株U2OS、SAOS2、ZOS及成骨细胞hFOB 1.19进行差异蛋白质组学分析，通过western blot及免疫荧光发现eEF1A2在8株骨肉瘤细胞中均呈现过表达状态，在化疗耐药株及高转移细胞株中过表达现象尤为明显。在前期研究的基础上通过原代培养、成球、化疗诱导富集TSCs，利用流式细胞仪分选CD117+/Stro-1+骨肉瘤TSCs；采用Western Blot、Real-time PCR、免疫荧光及功能实验等，明确eEF1A2与骨肉瘤TSCs的关系，并发现其与骨肉瘤的发生及转移存在密切联系；通过细胞、动物模型，验证eEF1A2在骨肉瘤TSCs成骨向分化及发生转移中的作用。通过表达谱芯片、生物信息学分析、基因转染、双荧光素酶报告基因实验等方法，发现eEF1A2通过SREBF1上IRES的活性调控SREBF1的表达，并重塑胞内脂质和胆固醇代谢平衡，从而调控骨肉瘤细胞干性，影响其发生发展的分子作用机制；体外实验发现通过SREBF1抑制剂Fatostatin抑制eEF1A2-SREBF1轴后可抑制肿瘤细胞的迁移和侵袭能力。上述结果为骨肉瘤的治疗提供潜在分子靶标，为改善骨肉瘤的整体临床效果提供理论依据及实验基础。