USP14抑制β-catenin泛素化降解促进肺腺癌恶性增殖及其分子机制的研究

The ubiquitin proteasome system has been implicated in tumorigenesis. Lung adenocarcinoma is one of the most common cancer in the world in terms of the high incidence and poor outcome. USP14 gene, which codes for one kind of deubiquitinating enzyme, was found overexpressed in both lung adenocarcinoma cell lines and postoperative lung adenocarcinoma specimens in our previous experiments. We also found that the elevated level of USP14 was associated with enhancement of proliferation of cancer cells. In this study we are going to explore the effects of endogenous USP14 on proliferation, apoptosis, cell cycle and invasive ability of lung adenocarcinoma cells both in vivo and in vitro. We are also trying to reveal the underlying mechanisms of the effects of USP14 on tumor cells. The degradation of ubiquitinated β-catenin has recently been proved to be inhibited by a series of deubiquitinating enzymes. Since the expression of β-catenin protein was found decreased along with USP14 gene interference, we will explore the correlation between the elevated level of USP14 expression and β-catenin accumulation in lung adenocarcinoma cells. By analysis of large sample of postoperative lung adenocarcinoma specimens we will try to find the possible correlation between USP14 expression and specified clinical features of the patients, including TNM staging and the outcome of the patients. We believe that through this study we can get further understanding on the biological characteristics of lung adenocarcinoma and the significance of post-translational modification on tumorigenesis and development.

研究肺癌的发生、发展机制，从中寻找早期诊断、预后判断的指标及新的治疗靶点始终是肺癌研究领域的热点。泛素/去泛素化系统的调节失衡与肿瘤的发生发展密切相关。基于前期在肺腺癌细胞株及手术标本中对去泛素化酶USP14的检测结果，以及发现USP14可能促进肺腺癌细胞株增殖的基础上，本课题拟采用基因干预技术，进一步从体内和体外两个层面明确内源性USP14对肺腺癌细胞恶性表型的影响；根据前期的USP14底物蛋白筛选结果，以β-catenin作为其主要靶蛋白，通过实验设计验证USP14通过对β-catenin的去泛素化调控而促进其在肺腺癌细胞内稳定表达，最终促进细胞增殖的分子机制。并检测300例肺腺癌临床样本，分析USP14的表达与肺腺癌患者病理学分级及预后等的相关性。希望通过本研究，对蛋白翻译后修饰在肿瘤发生发展中的作用提供新的认识，并为寻找肺腺癌诊断和预后判断指标及干预靶点奠定基础。

研究肺癌的发生、发展机制，从中寻找早期诊断、预后判断的指标及新的治疗靶点始终是肺癌研究领域的热点。泛素/去泛素化系统的调节失衡与肿瘤的发生发展密 切相关。基于项目前期在肺腺癌细胞株及手术标本中对去泛素化酶USP14的检测结果，以及发现USP14可能促进肺腺癌细胞株增殖的基础上，本课题采用基因干预技术，进一步从体内和体外两个层面明确内源性USP14对肺腺癌细胞恶性表型的影响，分析USP14的表达与肺腺癌患者病理学分级及预后等的相关性。希望通过本研究，对蛋白翻译后修饰在肿瘤发生发展中的作用提供 新的认识，并为寻找肺腺癌诊断和预后判断指标及干预靶点奠定基础。.本项目执行情况良好，严格按照原计划（No. 81201850）进行，原计划的研究内容均圆满完成。现将研究结果总结如下：.我们通过对多种肺癌细胞株及小样本肺腺癌患者手术标本进行mRNA表达谱芯片检测发现，多种非小细胞肺癌细胞株内USP14基因及蛋白的表达水平均有所增强，以肺腺癌细胞株最为明显，且USP14在肺腺癌组织中呈强阳性表达，且USP14高表达而生存分析提示USP14高表达组病人预后不佳。提示USP14在肺癌中发挥重要作用。我们的结果还发现，USP14可能通过Wnt/β-catenin信号通路参与肺癌的发生发展过程。至此已证明USP14抑制β-catenin泛素化降解，从而促进肺腺癌恶性增殖。此外，我们还应用此基金建立的一些实验体系研究了部分原计划没有列入的工作：通过生物信息学的方法和实验发现miR-4782-3p能够抑制USP14 mRNA水平的表达，进而提出miR-4782-3p/USP14/β-catenin信号通路可能参与对肺腺癌发生发展的调控的科学假说。通过细胞和动物实验，对上述推测加以验证，发现在肺腺癌细胞株及组织中，miR-4782-3p的表达是下降的，miR-4782-3p和USP14的表达呈负相关，而与肺癌患者的预后呈正相关。细胞实验中发现，miR-4782-3p和USP14之间存在双向抑制作用，USP14可部分逆转miR-4782-3p对肺癌细胞株功能的影响。标注本基金资助发表SCI论文2篇，另接收出版中1篇；拟继续发表基金相关SCI论文1篇。总之，本项目圆满完成。