TRAPPC4与ERK2作用的精细定位及其促进胃癌发生发展机制的研究

ERK/MAPK signal pathway regulates multiple cellular processes. How do ERK/MAPKs participate in the development of gastric cancer is one of hot spots nowadays. Scaffold proteins modulate the activity of ERKs via protein-protein interaction. Our study indicated that the expression of TRAPPC4 is upregulating in gastric cancer progressing, and it could distinctly interact with ERK2 on Golgi apparatus via its conserved Longin domain.TRAPPC4 could regulate the activity and spatial distribution of ERK2 in the way of function as a scaffold protein via interacting with MEK1 simultaneously.What's the accurate amino sites on which TRAPPC4 and ERK2 binds to each other and what's the exact subcellular spatial location that TRAPPC4-ERK2 interaction occurs? How TRAPPC4 promotes gastric cancer progessing in vivo and the relationship with clinicopathological characteristics and prognosis of gastric cancer patients? We attempt at explaining these questions by means of bioinformatics predicting,Co-IP, immunofluorescence,Fluorescence resonance energy transfer(FRET) assay,transfecting WT and mutant TRAPPC4 clones in vivo and in vitro， and establishing primary gastric cancer model using gene knocked-out mice, for the purpose of discovering and establishing molecular targets of prevention and therapy in gastric cancer.

ERK/MAPK 信号通路可调节多种细胞生命活动，研究胃癌的发生发展中该通路的调控机制是当前热点之一。目前认为脚手架蛋白在ERK活性的调控中发挥了重要的作用。前期研究发现运输蛋白微粒复合体TRAPPC4在胃癌发生过程中中表达逐渐上升。它与ERK2在高尔基体上直接作用，且是通过其LDc结构域与ERK2结合；TRAPPC4还可调节ERK2活化及空间分布，进一步发现它是通过同时招募MEK1从而发挥了ERK2脚手架蛋白的功能。但TRAPPC4与ERK2作用的精细定位如何？TRAPPC4在体内促使胃癌发生发展的机制及其与胃癌患者临床预后的关系亦尚未明确。为此本研究将通过生物信息学预测、Co-IP、免疫荧光、荧光能量共振转移（FRET）、体内/外转染野生型和突变型TRAPPC4、基因敲除鼠建模等方式，结合临床预后解答上述科学问题，为进一步发现和确立胃癌分子预防和治疗靶点提供实验依据。

1. SynbindinΔIEC小鼠构建：以C57BL/6N小鼠为背景鼠，利用KO First技术构建Synbindin KO First小鼠后，再依次与Fliper小鼠、Villin-Cre小鼠配繁，获得消化道特异性敲除Synbindin小鼠（SynbindinΔIEC小鼠）。.2.利用小鼠肠炎相关性结肠癌（colitis-associated cancer，CAC）模型，用AOM/DSS处理6-8周的肠上皮Synbindin特异性敲除小鼠（SynbindinΔIEC）及同窝对照小鼠（Synbindinflox），结果显示相比于对照Synbindinflox小鼠，SynbindinΔIEC小鼠结肠肿瘤发生率明显减少，肿瘤体积缩小，相应的肿瘤负荷明显降低，与我们既往的体外实验发现Synbindin可以促进胃癌细胞和结肠癌细胞的恶性生物学表型一致。.3.SynbindinΔIEC小鼠中降低的结直肠肿瘤发生发展可能与该小鼠中结肠上皮细胞的凋亡增加（TUNEL实验，WB检测分离的肠上皮细胞（IECs）中Bax和Bcl2蛋白水平），增殖减少有关（免疫荧光Ki67染色）。.4.SynbindinΔIEC小鼠与Synbindinflox在肠道菌群上有明显差异。.5. SynbindinΔIEC小鼠结肠正常肠上皮及肿瘤组织中ERK通路活性降低。.另外，证明了lncRNA -GClnc1的高表达与胃癌的发生、转移、肿瘤大小和病人预后不良呈正相关性，其作为脚手架分子结合并介导组蛋白甲基转移酶复合物的重要成员WDR5和组蛋白乙酰化酶KAT2A的相互作用及其对靶基因（以SOD2基因为代表的）的转录修饰，进而影响胃癌的细胞生物学功能（Cancer Discovery 2016）；得到同期编者评述。