羧基端SEA组件自酶切介导MUC3调控结肠癌EMT的分子机制研究

MUC3 is a typical membrane-tethered mucin expressed in gastrointestinal tract and epithelial cancers, which exerts a pivotal biological role, including cell signalling, cell proliferation and tumour progression or cell polarity. We have found that the carboxyl-terminal domain of MUC3 is posttranslationally autoproteolysed at the SEA module. The autoproteolysis at the SEA module of human MUC3 carboxyl-terminal domain modulates its functional composition, for example, regulating the epithelial-mesenchymal transition (EMT) in colon cancer cells, but the mechanism is largely unknown. Based on the our previous study, we postulate that the autoproteolysis at the SEA module of human MUC3 carboxyl-terminal domain leads to the interaction between the epithermal growth factor(EGF)-like motif flanked beside SEA module and ErBb2, one of the members of EGF receptor(EGFR), and further phosphorylation of ErBb2 at the different tyrosine sites, the phosphorylated ErBb2 leads to the activation of Wnt/β-cateinin siganaling, the translocated β-cateinin transcriptionally regulated the genes involved in EMT of colon cancer cells, and finally induced the occurrence of EMT. The study is planed to confirm the interaction between the EGF-like motif flanked beside SEA module and ErBb2, one of the members of EGFR, and further phosphorylation of ErBb2 at the different tyrosine sites due to the autoproteolysis at the SEA module of human MUC3 carboxyl-terminal domain using the specific antibodies for detecting different tyrosine phosphorylated ErBb2 and Western blotting, the quantitation of the expression levels of different Wnt signaling cascade (protein and mRNA) is used to confirm the activation of Wnt signaling cascade due to MUC3 Self-cleavage induced phosphorylated ErBb2, the transcriptional regulation of EMT-related genes by translocated β-cateinin is studied by the traditionally transcriptional regulation experiments, thus, to determine the molecular mechanism(s) of MUC3 Self-cleavage induced EMT occurrence in colon cancer cells. Hence, we will reveal the exact molecular mechanism underlying autoproteolysis modulating the biological behaviour of colon cancer cells, and thus offer an important clue for the understanding of colon cancer carcinogenesis.

MUC3是具重要生物学功能的膜结合型粘蛋白，我们发现MUC3羧基端翻译后经历SEA组件自酶切，而自酶切介导了MUC3调控结肠癌细胞EMT，但其分子机制不清。我们基于前期研究，提出MUC3羧基端SEA组件自酶切介导MUC3通过SEA组件侧翼EGF样结构域与EGFR家族的ErBb2结合并使其磷酸化，激活Wnt信号，调控EMT靶基因表达进而启动EMT的假说。本研究拟在细胞水平和动物模型水平，用不同酪氨酸位点磷酸化ErbB2抗体和免疫共沉淀实验证实MUC3通过EGF样结构域致ErbB2磷酸化，激光共聚焦、免疫印迹等检测Wnt信号相关蛋白证实ErbB2磷酸化激活Wnt信号，转录调控实验证实核转位的β-catenin调节EMT靶基因表达从而启动EMT。本课题揭示MUC3羧基端 SEA组件自酶切生物学意义及其介导MUC3调控结肠癌细胞EMT的分子机制，对认识MUC3的生物学功能和结肠癌发生机制具理论意义

MUC3是具重要生物学功能的膜结合型粘蛋白，其细胞外区域包括EGF 样结构域、SEA 组件等。后者是多种生物蛋白质分子内的保守序列，作为蛋白质翻译后修饰的重要形式。我们发现MUC3经历SEA组件自酶切介导了MUC3调控结肠癌细胞EMT及影响细胞生物学行为，但其生物学意义及分子机制不清。我们基于前期研究，发现MUC3羧基端SEA组件自酶切介导MUC3通过SEA组件侧翼EGF样结构域与EGFR家族的ErBb2结合并使其磷酸化，激活Wnt/β-catenin信号通路，调控EMT靶基因表达进而启动EMT，从而改变结肠癌细胞生物学行为。本研究在细胞水平，用不同酪氨酸位点磷酸化ErbB2抗体和免疫共沉淀实验证实MUC3通过EGF样结构域致ErbB2磷酸化，流式细胞术、激光共聚焦、免疫印迹等检测Wnt信号相关蛋白证实ErbB2磷酸化激活Wnt信号，揭示了MUC3羧基端 SEA组件自酶切生物学意义及其介导MUC3调控结肠癌细胞EMT的分子机制，我们进行了以下研究并取得相应重要结果：. 1. 成功构建并鉴定MUC3羧基端结构域的真核表达载体（p20）、酶切位点（LSKG/SIVV）中的S（丝氨酸）定点突变为A（丙氨酸）表达载体（p20S/A）；并构建相应稳定表达细胞株；V5抗体进行免疫荧光定位发现目的蛋白P20准确定位至细胞膜。. 2.免疫荧光及CO-IP实验证实了MUC3羧基端SEA组件自酶切通过侧翼EGF样结构域与EGFR家族成员ErbB2的结合；并促进ErbB2的Tyr1248位点磷酸化。. 3.明确了MUC3羧基端SEA自酶切激活wnt信号通路，并促进β-catenin蛋白向细胞核内转移。. 4.阐明MUC3 羧基端自酶切致ErbB2 磷酸化激活Wnt 信号通路，介导MUC3 调控结肠癌EMT，导致细胞生物学行为及EMT 标志的改变。. 以上结果证实了：MUC3 SEA 组件在LSKGSIVV 基序的G（甘氨酸）和S（丝氨酸）之间发生没有蛋白酶介导的自酶切后活化EGF 样结构域,，EGF 样结构域作为配体与ErbB2（EGFR）相互作用，导致ErbB2 磷酸化，后者激活Wnt/β-catenin信号通路，介导MUC3 调控结肠癌EMT及生物学行为。上述研究对认识MUC3的生物学功能和结肠癌发生机制具重要理论意义。