CPT1C调节乳头状甲状腺癌脂肪酸氧化代谢的机制及生物学意义研究

Papillary thyroid cancer (PTC), the most common type of thyroid carcinoma, has an impressive propensity for lymphatic spread. While it is well recognized that activating B-RAFV600E is the most prevalent mutation, understanding of the complete range of pathways involved in its pathogenesis is incomplete.Our earlier research, with metabonomics analysis （published in Mol Biosystmes ,2011） and clinical specimens check, found that fatty acid β-oxidation was promoted and brain type of carnitine palmitoyl-transferase 1 (CPT1) isozymes, CPT1C，was up-regulated. What is more, activating B-RAFV600E could induce CPT1C . In this project, we would like to confirm and partly clarify the significance of CPT1C up-regulation toward lipid metabolism and carcinogenesis under metabolic stress. Besides, it is to be confirmed in this project that whether B-RAFV600E mutation induce CPT1C expression through abating the microRNA-370, which negatively regulates CPT1C. We believe that implementation of this project will help to understand the molecular mechanism of thyroid carcinogenesis as well as provide clues for development of novel intervention against thyroid carcinomas.

乳头状甲状腺癌（PTC）是最常见的甲状腺癌，部分会侵犯淋巴结，产生一系列严重的症状和体征。B-RAFV600E突变是PTC主要的遗传学特征,但PTC的发病机制仍不清楚。近期，脂代谢异常与肿瘤发生发展的关系受到关注。申请者对PTC的血清和组织代谢小分子谱的研究显示：PTC的脂肪酸β－氧化（该过程的限速酶为肉碱脂酰转移酶I，CPT1）加速(Mol.Biosystems, 2011)，而原本表达于脑部的CPT1C亚型在PTC组织中表达显著上调，课题组的结果还提示B-RAFV600E可上调CPT1C。本项目拟研究（1）PTC癌组织中CPT1C的上调对PTC的脂肪代谢和恶性表型起何作用，有何意义；（2）PTC癌组织中CPT1C异常升高的分子机制是什么？包括是否与B-RAF经由miR-370调节有关系？阐明以上问题，有助全面认识PTC的分子发病机制，有望为研发新的治疗手段提供有效靶标。

脂肪酸氧化被认为在肿瘤细胞恶性表型中起一定作用。项目组之前的研究结果显示脂肪酸氧化的限速酶CPT1C可能在乳头状甲状腺癌的恶性转归中起一定作用。本项目中，首先应用transwell 实验检测转染CPT1C后的PTC细胞迁移能力的变化，结果显示过表达组CPT1C的转移能力比正常组的要强。细胞划痕实验检结果也显示过表达组的CPT1C比正常组的转移能力强。在KTC-1及B-CPAP两珠乳头状甲状腺癌细胞株利用小RNA干扰技术，干扰CPT1C进而观察CPT1C对对细胞周期和凋亡的影响，实验结果显示CPT1C干扰组的凋亡的比率明显上升和对细胞周期S期影响明显。结果还显示：CPT1C基因可以有保护缺氧应激的作用，提高肿瘤细胞生存率。.利用小RNA干扰技术，干扰CPT1C进而观察CPT1C对PTC在metabolic tress条件下细胞存活影响，实验结果显示低糖应激中干扰处理组的细胞株的存活状态明显低于对照组。接下来课题组用B-CPAP乳头状甲状腺癌细胞株利用小RNA干扰技术和质粒的瞬转技术，分别干扰CPT1C和过表达CPT1C, 低氧应激条件下干扰处理组的细胞株的存活状态明显低于对照组，过表达处理组的细胞株的存活状态明显高于对照组。在机制研究方面， 发现在低糖应激的处理下乳头状甲状腺癌细胞中AMPK与P53都表达量都有增加的现象的产生，显示在乳头状甲状腺癌中存在AMPK-ACC-CPT1C该信号通路的调节。项目的实施丰富了对PTC的发病机制的认识，为研发干预手段提供了线索。