CPEB4调控USP1/ID2和CAV1表达重编程诱导骨肉瘤生长发育的作用及机制研究

In clinic, Osteosarcoma that presents a multiplex differentiation abilities and kinds of subtypes, was regarded as a differentiation disease caused by disruption of osteoblast differentiation from mesenchymal stem cells(bMSC). In the last National Natural Science project, we found that CPEB4 expressed highly in osteosarcoma cells and tissue. Resent researches described that CPEB4 mediated translational control of many genes which caused growth and progression of tumor and that USP1/ID2 and CAV1 respectively play the key role in the beginning and development of osteosarcoma. Therefore, whether inhibition CPEB4 could block the growth and development of Osteosarcoma? Further experiments show that CPEB4 knockdown changes USP1 and CAV1 protein expression, without changes of mRNA level. Consequencely,we hypothesized that CPEB4 translationally control the activity of USP1 and CAV1,and induce multiplex differentiation and development of osteosarcoma. The project aims to clarify function and mechanism of CPEB4 involving in the osteosarcoma iniation and progression at the level of molecular,tissue and animal model using primary osteosarcoma cells, osteosarcoma cell lines, osteogenic cells and bMSCs. Using transgenic Mice Model asseses the side effects of inhibition CPEB4; Combing affymetrix microarray analysis and RIP-seq,according to the regulation of USP1 and CAV1 by CPEB4, acquires more CPEB4 regulated genes, and disclose the new reprogram of genes leading to normal cell transformation, which could provide a whole new stratege for osteosarcoma targeting therapy.

临床上骨肉瘤呈现多向分化和多种亚型，它是间充质干细胞（bMSC）成骨分化紊乱疾病。在国科基项目中，发现CPEB4在骨肉瘤细胞和组织中高表达；最近研究显示CPEB4在翻译水平调控肿瘤基因而诱导其生长发育，USP1/ID2和CAV1分别在骨肉瘤起始和发展中起重要作用。那么，干扰骨肉瘤中CPEB4，能否靶向阻断其诱导的肉瘤生长和再发育？进一步实验显示抑制CPEB4影响USP1和CAV1蛋白表达，而不影响mRNA表达。据此，我们假设CPEB4在翻译水平调控着USP1和CAV1，通过表达重编程来诱导骨肉瘤的多向分化和再发育。本项目拟通过原代骨肉瘤细胞、细胞系、成骨细胞及bMSC,从分子、组织及动物模型阐明CPEB4在骨肉瘤起始及再发育中的作用及机理；转基因小鼠评估抑制CPEB4功能效应，表达谱芯片与RIP-seq技术相结合，揭示CPEB4调控细胞恶变的基因表达重编程新机制，为骨肉瘤靶向治疗开启新途径

骨肉瘤是全身骨骼系统最常见的恶性肿瘤，其恶性程度高，危害大，血行转移较早且肺转移率较高。近年来，骨肉瘤的五年生存率大幅度提高到60%左右，但仍有40％左右的患者最终不可避免地发生肺转移。CPEB4作为CPEB（多聚腺苷化元件结合蛋白）家族中的一员，它能够促进多聚腺苷酸化诱导的翻译过程，并且能够介导干细胞发育、细胞分化和细胞衰老等过程。近几年来被发现在肿瘤发展过程中对基因转录后调节起关键作用。本研究旨在探讨CPEB4在维持骨肉瘤中生物特性中的作用及机制，以及CPEB4对发展新的靶向治疗的重要临床意义。本研究中，首先我们证实了CPEB4在多种骨肉瘤组织和细胞中高表达，进一步通过IHC验证CPEB4与骨肉瘤患者的预后具有显著地相关性。我们发现CPEB4在骨肉瘤成瘤方面发挥重要作用，其在骨肉瘤细胞和组织中的表达量与体内成瘤能力呈正相关；同时CPEB4在骨肉瘤的分化、耐药等方面起着重要作用，靶向抑制CPEB4可抑制骨肉瘤的生长及增加化疗药物敏感性；同时CPEB4可以抑制骨肉瘤细胞的分化能力。为了探讨CPEB4发挥作用的机制，我们利用肿瘤相关通路活性筛选及RIP-seq，筛选出GSK-3β/NF-κB信号通路为CPEB4直接调控的下游靶基因。进而明确CPEB4通过调控GSK-3β/NF-κB信号通路发挥癌基因的作用。