TGF-β/Akt/Smad介导的上皮间质转化在电离辐射增加肿瘤细胞转移潜能中的作用

Metastasis is one of the main reasons for the failure of cancer therapy, and it is also involved in radiotherapy. It has not been reported whether radiotherapy induce the metastasis of cancer cells directly. In previous studies, we concluded that ionizing radiation could promote metastatic potential of cancer cells through epithelial-mesenchymal transition (EMT), and the expressions of transforming growth factor-beta (TGF-β) were increased remarkably. However, cancer cells need to be irradiated many times continuously for several weeks in the course of radiotherapy, and the changes of EMT are still unknown in this process. Moreover, as an important trigger of EMT, what role of TGF-β in radiation-incuced EMT, and what about the transduction and crosstalk of its downstream signal, both of which are need to be clarified. In further study, some cancer cell lines originated from different human organs will be irradiated with X ray which come from conventional fraction-dose and times in clinical radiotherapy, and the changes associated with EMT will be observed, including morphology, EMT markers and functional indexes. Then the expressions of TGF-β and its downstream molecules related with Smad and Akt signals will be detected by immunological methods, and the effects and relationship between them in radiation induced-EMT will be analysed by some special approaches. This study will provide solid experimental foundation for explaining the relationship between ionizing radiation and tumor metastasis and the associated mechanisms. Meanwhile, it will provide theoretical evidence for the development of molecule-targeting agents.

转移是肿瘤治疗，包括放射治疗失败的主要原因之一，放疗是否能直接促进肿瘤细胞发生转移，尚未见报道。我们前期发现，单次常规分割剂量的电离辐射可以通过诱导离体肿瘤细胞发生上皮间质转化（EMT）进而增加其转移潜能，同时，细胞转化生长因子-β（TGF-β）的表达也明显升高。然而，放射治疗是连续几周的多次照射过程，该模式下肿瘤细胞EMT的变化规律如何，目前尚不清楚。此外，作为EMT的重要诱导因子，TGF-β在电离辐射诱导的EMT中扮演什么角色，其下游信号又是如何传递和发生交互作用的，均需进一步阐明。本课题将模拟临床放疗参数，照射不同组织来源的肿瘤细胞，从形态学、标志物和功能学角度观察EMT的变化规律，再运用免疫学方法检测TGF-β及下游Smad、Akt信号分子的改变，并通过多种干预手段分析它们的作用及相互关系。本研究将为明确放疗和肿瘤转移的关系及机制提供实验基础，为相关分子靶向药物的研发提供理论依据。

转移是肿瘤治疗，包括放射治疗失败的主要原因之一，放疗是否能直接促进肿瘤细胞发生转移，尚未见报道。我们前期发现，单次常规分割剂量的电离辐射（IR）可以通过诱导离体肿瘤细胞发生上皮间质转化（EMT）进而增加其转移潜能，同时，细胞转化生长因子-β（TGF-β）的表达也明显升高。然而，放射治疗是连续几周的多次照射过程，该模式下肿瘤细胞EMT的变化规律如何，目前尚不清楚。此外，作为EMT的重要诱导因子，TGF-β在IR诱导的EMT中扮演什么角色，其下游信号又是如何传递和发生交互作用的，均需进一步阐明。.本课题首先模拟临床放疗参数，建立放射抵抗肿瘤细胞模型，从形态学、分子标志物、功能学水平观察电离辐射诱导后肿瘤细胞EMT的发生规律。结果显示，IR诱导后，肿瘤细胞发生了EMT相关事件：间质表型的形成，上皮标志物表达的减少和间质标志物表达的增加，细胞迁移和侵袭能力的增加。.其次，蛋白水平检测TGF-β及下游信号Smad3、Akt的表达，结果显示，IR诱导后，肿瘤细胞TGF-β的表达及下游信号分子Smad3、Akt的磷酸化水平明显增加，其中A549细胞变化趋势最明显，其将作为后续研究的对象。.最后，观察TGF-β/ Akt/Smad3通路在IR诱导肿瘤细胞EMT过程中的作用。结果显示：应用TGF-β通路抑制剂，下游p-Smad3和p-Akt表达均下降；加入Akt通路抑制剂后，p-Smad3和p-Akt水平均降低；而引入Smad3的SiRNA处理后，p-Smad3表达降低，而p-Akt水平无明显下降；每种处理因素加入后，A549细胞上皮标志物E-Cadherin表达升高，间质标志物N-Cadherin表达降低，划痕伤口愈合面积及Transwell穿膜细胞也明显减少；之后Co-IP实验证实Smad3和Akt存在相互作用。.通过以上研究，可以得出结论：IR能够促进离体肿瘤细胞EMT的发生，TGF-β/ Akt/Smad3信号在其中均扮演着重要的角色。.本研究将为明确放疗和肿瘤转移的关系及机制提供实验基础，为相关分子靶向药物的研发提供理论依据。