重组人食管癌相关基因4蛋白在食管癌中的抑癌作用和机制研究

Esophageal cancer related gene 4 (ECRG4) is a candidate of tumor suppressor genes, which is closed associated with tumorigenesis and prognosis. Until now, there is few studies on the tumor-suppressing function and molecular mechanism of ECRG4 protein. The applicant's preliminary research results found that recombinant human ECRG4 protein inhibited tumor cells proliferation in vitro, and that ECRG4 protein interacted with transmembrane protease serine 11A (TMPRSS11A) protein. In this study, the ECRG4 expression vector is constructed and the recombinant human ECRG4 protein is purified. We research the tumor-suppressing function of recombinant human ECRG4 protein in vitro and in vivo through colony formation experiment in flat and soft agar, flow cytometry cell cycle analysis, cell proliferation/ migration/ invasion/ adhesion assay, tumor growth in nude mice experiment, et al. And the TMPRSS11A protein deletion mutant is constructed to research the protein interaction with ECRG4 and key molecular sites through GST-pull down assay, laser-scanning confocal microscopy analysis, and Co-immunoprecipitation experiment. Finally, we try to provide experimental data for clarifying the tumor-suppressing function and molecular mechanism of ECRG4 in esophageal carcinoma, and to lay experimental foundation for recombinant ECRG4 protein as candidate biotherapy drug in esophageal carcinoma.

食管癌相关基因4（ECRG4）与肿瘤发生发展和预后密切相关，是候选抑癌基因。然而，ECRG4蛋白的抑癌作用和机制研究甚少。申请人研究发现，重组ECRG4蛋白体外抑制肿瘤细胞增殖，与跨膜丝氨酸蛋白酶11A(TMPRSS11A)之间存在蛋白相互作用。本课题构建ECRG4表达载体，纯化重组ECRG4蛋白，通过平板集落和软琼脂克隆形成实验、流式细胞周期分析、细胞增殖/迁移/侵袭/粘附实验、裸鼠成瘤等体外和体内功能实验，研究重组ECRG4蛋白在食管癌中的抑癌作用；另外，构建TMPRSS11A蛋白缺失突变体，通过GST-pull down实验、免疫共沉淀、激光聚焦荧光共定位分析，探讨ECRG4与TMPRS11A之间的相互作用和其关键分子位点。本研究探索ECRG4在食管癌中的抑癌作用和机制，为重组ECRG4蛋白作为食管癌潜在生物治疗药物提供实验依据。

本课题构建人食管癌相关基因4（esophageal cancer related gene 4，ECRG4，also called C2ORF40）过表达和低表达慢病毒载体。同时成功获得高纯度可溶性重组人ECRG4蛋白。 研究发现重组ECRG4蛋白体外抑制肿瘤细胞增殖，诱导细胞周期 G1期阻滞；体内抑制裸鼠移植瘤生长。免疫共沉淀实验结果表明 ECRG4蛋白和TMPRSS11A蛋白相互作用。 .利用TMT定量蛋白质组学分析发现：肿瘤细胞中ECRG4过表达上调310个蛋白，下调175个蛋白，包括CDK4、PLOD2、PLOD1、PLK1、SNIP1、P65等。 临床食管癌配对组织芯片标本免疫组化结果验证，ECRG4蛋白表达与CDK4、PLOD2、PLOD1、PLK1具有相关性；Western blot 检测，ECRG4蛋白过表达下调CDK4、PLOD2、PLOD1、PLK1，ECRG4蛋白低表达上调CDK4、PLOD2、PLOD1、PLK1。ECRG4过表达增加细胞凋亡，抑制细胞增殖；ECRG4低表达促进细胞生长。.研究还发现ECRG4通过调控SNIP1与P65结合介导食管癌细胞凋亡。ECRG4过表达下调SNIP1、P65、BCL2、BCL-XL，ECRG4低表达上调SNIP1、P65、BCL2、BCL-XL，SNIP1低表达下调P65、BCL2、BCL-XL，ECRG4和SNIP1 同时低表达下调BCL2和BCL-XL蛋白表达程度降低。ECRG4低表达抑制细胞凋亡，SNIP1低表达促进细胞凋亡，ECRG4和SNIP1 同时低表达促进细胞凋亡程度降低。ECRG4低表达促进细胞增殖，SNIP1低表达抑制细胞增殖，ECRG4和SNIP1 同时低表达抑制细胞增殖程度降低。免疫共沉淀实验结果表明ECRG4蛋白过表达降低SNIP1与P65结合稳定性。利用ECRG4过表达的食管癌细胞系建立裸鼠皮下瘤模型，ECRG4蛋白高表达抑制裸鼠移植瘤生长，组织标本Western blot检测，ECRG4高表达下调SNIP1、P65、BCL2、BCL-XL蛋白表达，Tunel染色分析ECRG4高表达促进肿瘤细胞凋亡。.本课题研究结果表明ECRG4是食管癌候选抑癌基因，调控SNIP1与P65等多个信号通路，在食管癌的发生发展中扮演了重要角色；重组ECRG4蛋白可以作为食管癌潜在的生物治疗药物