DEPDC1/MCAK/ERK正反馈环路促进肺腺癌细胞保护性自噬的分子机制与临床意义研究

DEPDC1 is one of the tumor-related genes that our group has been studying. In the early stage, we mainly focused on its role in the cell cycle. Recently, the role of DEPDC1 in the development of lung cancer has drawn our attention. After bioinformatics analysis and a series of pre-experimental exploration, we found that DEPDC1 expression in lung cancer tissues is higher than normal tissues, and is closely related with the prognosis. DEPDC1 and MCAK are mainly Co-localized in the nucleus in lung cancer cells. Co-immunoprecipitation analysis indicated that they are interacted. Exogenous overexpression of DEPDC1 in H1299 can promote cell autophagy and proliferation and the expression of phosphorylated ERK is Up-regulated. Thus, we proposed our academic guess "DEPDC1-MCAK interaction promote autophagy and proliferation through the regulation of ERK signaling pathway in lung cancer cells, thereby promoting the occurrence and development of lung cancer." We are ready to adopt a variety of molecular biology techniques to confirm the interaction of DEPDC1 and MCAK and analyze in-depth its role in the development of lung cancer and its clinical significance. Provide a theoretical basis for its molecular diagnosis and treatment of lung cancer.

前期我们集中于DEPDC1在细胞周期中的作用研究。最近，DEPDC1在肺腺癌发生发展中的作用引起我们的关注。经过生物信息学分析及一系列的预实验探索，我们发现：DEPDC1在肺腺癌组织中表达高于正常组织，且其高表达的患者预后较差；肺腺癌细胞中DEPDC1与MCAK存在共定位，免疫共沉淀初步分析二者存在互作；肺腺癌细胞A549中DEPDC1、MCAK外源过表达可促进细胞自噬与增殖，且磷酸化的ERK出现表达上调。另有研究报道ERK可促进MCAK的表达，且ERK2与DEPDC1、MCAK存在显著的正相关的关系。由此提出学术猜想：“DEPDC1与MCAK互作，通过正反馈调控ERK促进肺腺癌细胞保护性自噬（即通过自噬促进增殖、周期进程），从而促进肺腺癌的发生发展进程”。本课题拟在此基础上，证实DEPDC1/MCAK/ERK正反馈环路在肺腺癌发生发展中的作用及临床意义。

前期研究发现敲降DEPDC1后细胞出现多极纺锤体以及多个细胞核进而发展到细胞分裂异常，导致细胞凋亡。为了进一步拓展研究DEPDC1的功能，我们展开研究了DEPDC1在肺腺癌发生发展中的作用。免疫荧光实验分析发现肺腺癌细胞中DEPDC1与MCAK存在共定位，免疫共沉淀及分子对接模拟分析发现两个蛋白存在相互作用；肺腺癌细胞中DEPDC1、MCAK外源过表达可促进细胞自噬与增殖，且磷酸化的ERK出现表达上调。敲降DEPDC1或MCAK表达后，细胞自噬减弱，增殖减慢，平板克隆数减少；敲降DEPDC1或MCAK表达后，RNA-seq转录组测序分析发现差异表达基因可富集到调控ERK的相关信号通路如MAPK信号通路，RAS信号通路。将差异表达基因进行网络互作分析，发现潜在的处于下游的关键基因如PLK1、CDC6、MAPK11、SAMD3等基因；分析来源于TCGA及GEO数据库中的多种实体瘤的mRNA表达数据集，发现DEPDC1、MCAK在肺腺癌、肺鳞癌、结肠癌、乳腺癌、头颈癌组织中的mRNA表达水平显著高于正常组织，肺腺癌中与分级相关，且其高表达的患者预后较差。肺腺癌中DEPDC1、MCAK与参与自噬过程的ATG3、ATG4B、ATG4C、ATG5、ATG7、ATG12、ATG13、ATG101、ULK1等基因的mRNA表达水平存在不等程度的正相关的表达关系。综上，我们的研究发现肺腺癌细胞中DEPDC1与MCAK蛋白存在相互作用，两个蛋白参与肺腺癌细胞的自噬过程，调控ERK信号通路，进而调控肺腺癌细胞的增殖迁移等过程，从而促进肺腺癌的发生发展进程。本研究为评估DEPDC1作为肺腺癌的诊断或者靶向治疗的靶点提供理论基础。.项目期间我们利用TCGA数据库中肺腺癌数据集构建了肺腺癌基因加权共表达分子网络，得到几个在肺腺癌中高表达的分子网络和模块，针对其中的E2F2为核心的分子网络进行后续的实验验证。发现E2F2、B-MYB、FOXM1三个核心转录因子组成正反馈调控环，调控下游的靶基因PLK1、DEPDC1、CENPF、CENPA、CCNA2、BRIP1、BIRC5、AURKB等基因，进一步影响肺腺癌细胞的细胞周期进程、细胞运动以及AKT信号通路进而参与肺腺癌的进程。

内容获取失败，请点击重试