RanBP9调控骨肉瘤失巢凋亡的分子机制研究

The anoikis resistance of osteosarcoma is a key point in the process of its invasion and metastasis.So the molecular mechanism of anoikis in osteosarcoma remains a difficulty and hotspot problem. Our previous work revealed that the acquired ability of anoikis resistance in osteosarcoma cells played an important role in the process of metastasis.Imprinting gene TSSC3 participated in the process of anoikis resistance of osteosarcoma as a tumor supressor. Using yeast two-hybrid system, we firstly identified RanBP9 as a novel cellular protein involved in the regulation of TSSC3. As reported by other researches, the RunBP9 also participate in the process of apoptosis.But how the TSSC3 was regulated by RanBP9 was unclear. So in this study, we would investigate firstly: the biological functions of RanBP9 in osteosarcoma, especially in the process of anoikis resistance in osteosarcoma regulated by TSSC3. The relationship between RanBP9 and TSSC3 in the anoikis signals also will be investigated. Last, we will investigate the mechanisms of the interactions between TSSC3 and RanBP9, to determine their binding region and the stability effects of RanBP9 on TSSC3. Thus, we may found the possible mechanisms by which TSSC3 interacting with RanBP9 in the process regulating osteosarcoma anoikis, providing a theoretical basis for TSSC3 and RanBP9 as osteosarcoma therapeutic targets.

骨肉瘤抗失巢凋亡是其侵袭和转移过程中的关键事件。因此深入探讨其抗失巢凋亡的分子机制非常必要。我们前期研究发现，印迹基因TSSC3以抑癌基因参与调控骨肉瘤失巢凋亡，并通过酵母双杂交实验钓取了TSSC3新的相互作用蛋白RanBP9。RanBP9在骨肉瘤失巢凋亡诱导模型中具有与TSSC3一致的变化趋势和胞质共定位。但RanBP9在骨肉瘤中的作用，及RanBP9是如何通过与TSSC3相互作用而调控骨肉瘤失巢凋亡的机制目前尚不清楚。为此，本实验拟首先明确RanBP9 在骨肉瘤及失巢凋亡过程中的作用；并探讨RanBP9对 TSSC3调控的骨肉瘤失巢凋亡等生物学特性的影响以及二者在骨肉瘤抗失巢凋亡关键信号通路Src/PI3K/Akt中的相互作用关系；最后确定RanBP9与TSSC3的结合域。本项目的完成，将为深入阐明骨肉瘤抗失巢凋亡的分子机制提供新的认识和思路，并为改善骨肉瘤的诊治提供新靶点和新策略。

失巢凋亡抵抗是肿瘤发生远处转移过程中首要且关键的步骤，与患者的化疗药物抵抗及不良预后相关。本项目中，首先我们采用酵母双杂交系统，钓取与TSSC3发生相互作用的蛋白RanBP9，并进一步利用免疫共沉淀等鉴定两蛋白的相互作用，结果表明：TSSC3/RanBP9复合物在核酸及蛋白水平均存在相互作用；进一步实验中，我们利用构建突变载体发现介导TSSC3与RanBP9相互作用的结构域为RanBP9的SPRY以及TSSC3的PH结构域。其次，以骨肉瘤细胞及人骨肉瘤组织为研究材料，检测TSSC3与RanBP9在骨肉瘤中的表达及其与临床病理学参数的相关性，发现：TSSC3/RanBP9与骨肉瘤细胞的失巢凋亡抵抗呈负相关，与骨肉瘤的肺转移呈负相关。进一步利用相关分子生物学实验探讨TSSC3/RanBP9对骨肉瘤失巢凋亡和肺转移的影响和可能的分子机制，结果表明：RanBP9与TSSC3协同抑制了骨肉瘤的锚着依赖生长、促进其失巢凋亡（线粒体途径）并最终抑制了骨肉瘤的远处转移，该效应依赖于其对Src/PI3K/Akt通路的抑制作用。进一步研究表明RanBP9的SPRY结构域是介导该复合物上述生物学效应的关键区域。综上，我们的研究阐述了RanBP9和TSSC3复合物的形式协同促进失巢凋亡、抑制转移，RanBP9是一个脚手架蛋白，它增强了TSSC3/RanBP9/Src的结合，进而抑制Src的活性，抑制Akt通路，最终激活线粒体途径介导的失巢凋亡