基于体内外模型的Par3L调控结直肠癌发展及其与14-3-3相关分子机制的研究

Colorectal cancer (CRC) has gradually become one of the top three cancers in the world. Investigation on the mechanisms of CRC development and search for the molecular probe of CRCs, are urgent and significant for CRC diagnosis and clinical treatment. Previous studies indicate that: 14-3-3 synergistic Hippo signaling inhibits Yap transcriptional activity which regulates multiple tumor development; Par3 and its homolog-Par3L can bind to 14-3-3 respectively, while Par3-14-3-3 protein combination inhibits tumor metastasis. Contrast to loss of Par3 in many tumors, our previous observations show that the expression of Par3L is significantly increased in CRC tissues compared with adjacent non-tumor colon tissues, and Hippo pathway activity is attenuated in Par3L high expressed CRC tissues, monitored by ectopic expression of Yap target genes,moreover, Knockdowning Par3L inhibits both proliferation and migration of CRC cells. According to these results, we propose the hypothesis that Par3L-14-3-3 protein combination has biological function of promoting CRC tumorigenesis and metastasis through inhibiting Hippo pathway. In this project, based on in vitro and in vivo models of patient cancer samples, colorectal cancer cell lines and orthotopic xenograft in nude mice, we will employ cell biology methods, molecular biology technique, physiological and biochemical technology, to explore and clarify functions of Par3L regulating CRC development, and to explore the inhibitory of Par3L-14-3-3 protein combination on Hippo signaling and uncover the underlying mechanism. The results of this study will reveal mechanisms of CRC development, and provide molecular probe and target for CRC diagnosis and clinical treatment.

结直肠癌是世界前三大癌之一，探究其发病机理并寻找其分子探针，对其诊断治疗有重大科研和临床意义。已报道，14-3-3协同Hippo信号通路抑制Yap转录活性调控多种肿瘤发展，Par3及其同源蛋白Par3L都能与14-3-3结合，而Par3-14-3-3抑制肿瘤转移。与多种肿瘤缺失Par3相反，我们前期研究显示病人结直肠癌组织Par3L蛋白表达增高且Hippo信号通路失活、Yap转录活性上升，敲弱Par3L抑制结直肠癌细胞增殖和迁移。我们据此推断Par3L-14-3-3有抑制Hippo信号通路促进结直肠癌发展的功能。本项目拟基于临床病人样本、结直肠癌细胞系和原位移植肿瘤的体内外模型，采用细胞、分子生物学及生理生化技术探究和明确Par3L对结直肠癌发展的调控作用，厘清Par3L-14-3-3对Hippo信号作用并解析其机理，从而揭示结直肠癌发展分子机制、为临床诊断治疗提供分子探针和着力靶点。

前人临床数据、研究结论和前期研究结果提示Par3l可能调控结直肠癌发展，为明确其作用揭示其机理，本项目利用Crispr-cas9技术敲除了人结直肠癌细胞中Par3l基因、构建了Par3l过表达转基因小鼠。MTT、Transwell和克隆形成实验分别显示Par3l基因敲除后显著抑制了细胞增殖、迁移和形成克隆能力。质粒转染实验显示过表达Par3lWT可以显著恢复Par3l敲除细胞的增殖能力，而Par3lS746A点突变质粒则不能。裸鼠皮下接种实验显示，Par3l敲除显著下调了结直肠癌细胞在体内瘤体形成和增长活力，而在ApcMin/+鼠中过表达Par3l可显著缩短其生存时间。全mRNA测序分析结果显示Par3l敲除影响了调控细胞信号通路、细胞因子与其受体及细胞EMT和迁移相关基因的表达。QPCR数据显示裸鼠皮下瘤体中，Par3l敲除结直肠癌瘤体中Il20ra、Dcr3、c-Myc和Twist1 mRNA水平显著下降，而该瘤体的WB数据显示Par3l敲除显著下调了STAT3蛋白及其磷酸化的水平。综合以上结果提示Par3l能通过增强体外结直肠癌细胞增殖、迁移、克隆形成和在体内成瘤和增长活力而促进结直肠癌的发展，其机制可能主要通过与其746S关联的14-3-3蛋白互作，及通过调控细胞因子受体Il20ra、Dcr3和STAT3及其磷酸化水平促进c-Myc和Twist1表达。从而明确了Par3l体内外调控结直肠癌发展的作用并揭示了其调控新机制，为结直肠癌的诊断与治疗提供了新的潜在靶点和思路。