Cldn-7调控Intergrin/FAK信号通路参与大肠癌发生、侵袭转移的机制研究

Study on the role and mechanism of intergrin/FAK signaling pathway in tumorigenesis fields of many malignant tumors are popularly performed recently.Claudin-7 is a newly important gene of which is closed associated with the development,infiltration and metastasis of colorectal carcinoma(CRC).However,the modulation mechanism of Claudin-7 worked on the intergrin/FAK pathway has not been reported as for the literature. We previously found the interaction between Cldn-7 and intergrin α2 in intestine,as well as intergrin β1 did.Furthermore,the Cldn-7 protein expression level was associated with the colon cancer differentiation and metastasis.Cldn-7 expression will be upregulated and downregulated in the colon cancer cells using the gene transfection method in our study,to detect the effect on the intergrin/FAK pathway activity and downstream gene expression level and to observe its effect on the differentiation,apoptosis,invasion and metastasis of colorectal cancer cell.To explore the direct evidence of Cldn-7 effect on colon cancer growth using the establishment of the Cldn-7 conditional gene knockout mice, combined with clinical data of colorectal cancer.Moreover,we are to investigate the mode of action on intergrin/FAK pathway regulated by Cldn-7 and explore the molecular mechanism of Cldn-7 promotion on colon cancer invasion and metastasis.Theoretical and experimental basis will be provided in order to elucidate the mechanism.

Intergrin/FAK信号通路在恶性肿瘤发生发展中的作用机制是该领域的研究热点。Cldn-7是新近发现与大肠癌发生、侵袭转移密切相关的重要基因，其对intergrin/FAK信号通路的调控研究未见报道。我们前期研究发现肠道中Cldn-7和intergrinα2和β1相互作用，且Cldn-7蛋白表达与大肠癌分化和转移相关。本课题拟用基因转染和干扰技术分别上调和下调大肠癌细胞Cldn-7表达，检测其对intergrin/FAK通路活性及下游基因表达的影响，观察大肠癌细胞增殖、凋亡、侵袭转移能力变化；建立可诱导Cldn-7条件性基因敲除小鼠，结合临床资料，寻找Cldn-7基因影响大肠癌生长的直接证据；进一步用Co-IP、ChIP等技术研究Cldn-7调控intergrin/FAK通路的作用模式，探讨Cldn-7参与大肠癌发生、侵袭转移的分子机制，为阐明大肠癌发生发展机制提供理论和实验依据。

结直肠癌是全世界范围内常见的恶性肿瘤之一。其发病率和病死率逐年上升，对人类健康造成了严重威胁。现认为紧密连接蛋白Claudin-7是结直肠癌潜在的抑癌基因，由于其可作为结直肠癌的治疗靶点而逐渐受到重视。经过五年的研究工作，课题组初步获得了三个方面的研究成果：一、成功构建可诱导性肠道条件性Claudin-7基因敲除小鼠，并摸索出他莫昔芬可诱导出肠道腺瘤的合适剂量。对该小鼠模型进行肠道组织切片HE染色，发现小鼠肠上皮细胞明显脱落、极性丧失、并伴有显著的黏膜增厚和大量的炎性细胞（淋巴细胞、中性粒细胞、巨噬细胞）浸润，同时可观察到局部病理增生及肠腺瘤。该模型为首例Claudin-7可诱导肠道条件性基因敲除小鼠，成功避免了以往Claudin-7基因完全敲除后造成的出生后不久即死亡的弊端，可以在时间和空间上控制Claudin-7基因的敲除，在科研方面具有较强的学术价值。同时可诱导基因敲除小鼠也为研究人类大肠癌提供了动物模型，使在动物体内研究大肠癌的发生机制、抗肿瘤药物的应用成为了可能，具有较强的临床研究应用价值。二、课题组收集了多例大肠癌患者肿瘤组织样本及癌旁组织样本，利用免疫组化、Westernblot等技术进行Claudin-7及EMT相关基因表达的检测，发现Claudin-7在大肠癌中的表达较正常大肠组织降低，表达水平与大肠癌组织分化水平呈正相关，随分化程度的降低而降低。大肠癌组织中Claudin-7蛋白表达与E-cadherin表达呈正相关，与Vimentin、Snail-1表达呈负相关，提示Claudin-7可能参与大肠癌上皮-间质转化的发生。三、筛选高表达Claudin-7蛋白的细胞系HCT116，构建慢病毒干扰载体感染细胞进行该基因的敲降。利用Westernblot检测基因敲降效率后进行细胞功能学实验。Claudin-7干扰后，细胞功能实验显示大肠癌细胞HCT116的增殖及迁移能力增强。免疫荧光结果显示，敲降Claudin-7基因表达的HCT116细胞中Integrin-β1表达明显降低，CO-IP显示二者之间存在相互作用。大肠癌细胞实验研究进一步揭示了Claudin-7作为抑癌基因影响大肠癌发生发展的分子作用机制。