E2/E3泛素杂合酶UBE2O调控Mxi1泛素化降解促进肺癌放疗抵抗的作用机制研究

Radiochemoresistance is considered the main cause of local recurrence and distant metastasis in lung cancer. We have previously demonstrated that Mxi1 is downregulated and enhances the radiosensitivity in lung cancer. However, the mechanisms by which Mxi1 is regulated have not yet been fully understood. Using tandem affinity purification and immunoprecipitation technologies, our preliminary data show that E2/E3 hybrid ubiquitin-protein ligase UBE2O binds to Mxi1 and negatively controls the protein level of Mxi1. Functional assays show that UBE2O silencing enhances radiochemosensitivity in lung cancer cells. Therefore, we hypothesize that UBE2O interacts with Mxi1 and promotes the degradation of Mxi1 via ubiquitin-proteasome pathway, which ultimately results in Myc activation and promotes cell radioresistance in lung cancer. This study will elucidate how Mxi1 is controlled by UBE2O mechanistically and explore the role and clinical value of UBE2O in lung cancer，and may be provide insight into the novel molecular mechanism of radioresistance and a potential therapeutic and radiosensitization target for lung cancer.

放疗抗拒是导致肺癌局部治疗失败和远处转移的重要因素之一。本课题组既往研究发现Myc抑制蛋白Mxi1在肺癌中低表达并参与调控肺癌的放疗抵抗，但其调控机制尚未完全明确。申请人通过串联亲和纯化和免疫共沉淀的方法发现E2/E3 泛素杂合酶UBE2O可以与Mxi1相互作用且负向调控其蛋白水平，提示UBE2O可能是Mxi1的E3泛素连接酶。初步的功能学实验显示沉默UBE2O可增加肺癌细胞的放疗敏感性。因此我们推测：E2/E3杂合酶UBE2O通过与Myc抑制蛋白Mxi1结合，促进Mxi1的泛素化降解，从而激活Myc的转录活性，最终导致肺癌放疗抵抗。本研究将进一步从分子、细胞及动物水平探求UBE2O泛素化降解Mxi1最终参与肺癌放疗抵抗的机制，同时明确UBE2O在肺癌中的临床及预后意义。本研究的完成有望阐明肺癌放疗抵抗的新机制，同时也可为肺癌放射治疗提供新的治疗靶点。

放疗抵抗是导致肺癌局部治疗失败和远处转移的重要因素之一。本课题组既往研究发现Myc抑制蛋白Mxi1在肺癌中低表达并参与肺癌的放疗抵抗，但其上游调控机制尚未完全明确。本研究中，我们发现E2/E3泛素杂合酶UBE2O可以与Mxi1相互作用，并通过Mxi1的K46位点介导Mxi1泛素化降解，从而负向调控Mxi1的蛋白稳定性。进一步的机制探索显示UBE2O可以和E3泛素连接酶β-Trcp协同促进Mxi1的泛素化降解。功能学上我们发现在肺癌中敲低UBE2O或抑制UBE2O的活性均能在体内外发挥抑瘤和放射增敏效应，且这些效应主要依赖于UBE2O对Mxi1蛋白的降解。在肺癌组织中UBE2O蛋白高表达，其高表达提示肺癌患者预后不良，且UBE2O与Mxi1在肺癌组织中的表达呈负相关。因此，本研究揭示了UBE2O促进Mxi1泛素化降解并导致肺癌放疗抵抗的分子机制，提示靶向UBE2O/Mxi1通路可能是肺癌放疗增敏的理想策略。

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