E3泛素链接酶MKRN1在结直肠癌发生中的作用机制研究

Colorectal cancer (CRC) is one of the most common malignancies worldwide and a large amount of evidence indicates that deregulation of E3 ligase often results its progression and development. In previous study of mammalian spermatogenesis we identified a testis abundent E3 ligase MKRN1, further investigation found it highly expressed in clinical colorectal cancer samples. To explore the role of MKRN1 in colorectal tumorigenesis, we generated an MKRN1 knockout (KO) HCT-116 human colorectal cancer cell line as a research model. Compared to WT colorectal cancer cells, the colony forming efficiency and doubling time were obivious reduced in MKRN1 KO cells, which indicates that MKRN1 plays a critical role in colorectal tumorigenesis. In this project, we intended to enroll more clinical CRC patients to investigate the association between MKRN1 and various of clinical related outcomes of CRC and make clear of the identity of MKRN1 as a tumor related protein both in vitro and vivo. We will treat nude mice with MKRN1 recombinant protein or lentivirus stable cell lines to explore the role of MKRN1 in tumor progression. Furthermore, We ought to develop a tandem affinity purification (TAP) methods coupled with mass spectrometry and co-immunoprecipitation to detect the possible interaction proteins of endogenous MKRN1. We also wish to investigate the detail molecular mechanismsfor MKRN1in CRC progression, analysis its regulation, target substract and involving signal pathway both in vivoand in vitro. This research will provide important clue to discover new molecular target drug for CRC therapy.

结直肠癌是全世界范围内高发的恶性肿瘤，其发生发展与某些泛素连接酶的异常调节密切相关。本课题组在精子发生的机理研究中发现一个睾丸高表达的泛素连接酶MKRN1，进一步研究发现它在临床结直肠癌样本中高表达，通过体细胞基因敲除技术构建MKRN1-/-的结肠癌HCT-116细胞表现出明显的生长迟缓和克隆形成降低的能力，上述结果提示MKRN1可能参与调控结直肠癌的发生。本项目拟扩大临床结直肠癌样本明确MKRN1与结直肠癌病人各项临床指标的关系；制备MKRN1重组蛋白，通过穿膜侵袭和裸鼠成瘤等实验鉴定MKRN1肿瘤相关蛋白的身份；拟建立串联标签标记内源性MKRN1并结合免疫沉淀和质谱分析鉴定其相互作用蛋白，从细胞、动物和临床病人深入研究MKRN1在结直肠癌发生中的上游调控机制并寻找下游功能底物及其参与调节的主要信号通路，揭示其在结直肠癌中的作用及分子机理，为结直肠癌治疗的新分子药物靶标提供重要线索。