肉毒碱棕榈酰基转移酶1A(CPT1A)通过调控活性氧表达促进脱离基质的肝癌细胞在循环血液中存活的机制研究

Anoikis resistance is an important reason for tumor cells away from the extracellular matrix to occur tumor invasion and metastasis. Proper regulation of reactive oxygen species (ROS) is the key point to prohibit anoikis resistance, but the specific mechanism how to regulate still remains unknown. Carnitine palmitoyltransferase 1A (CPT1A) is a key enzyme to regulate lipid metabolism. We previously found that the accumulation of fatty acids and reducing of acetyl carnitine content in hepatocellular carcinorma (HCC) tissue，which significantly correlated to the degree of malignancy and recurrence of HCC, can be recognized as a good predictor of diagnosis and prognosis. Thus we speculate that there is abnormal expression of CPT1A in HCC which result in fatty acid accumulation then triggering ROS budding, finally to induce hepatoma cell anoikis resistance. The results of pre-experiment showed that ROS increased significantly after CPT1A overexpression in hepatoma cell, while their anti-apoptosis and clonogenic ability enhanced. In this study, we will clinically clarify the correlation between CPT1A expression level, recurrence and metastasis. Then CPT1A overexpression / silencing hepatocarcinoma cell line and conditional knockout mouse model were constructed to discover the correlation between CPT1A expression, ROS and anoikis resistance. Further, we will explore the mechanism of CPT1A promote hepatoma cell anoikis resistance and metastasis through regulating intrahepatoma ROS.Finally, we will repeat above hypothetical research in animal model to verify the experiments results.

肿瘤细胞脱离ECM后发生失巢凋亡抵抗是肿瘤侵袭转移的重要原因，活性氧（ROS）的有效调控是阻止细胞失巢凋亡抵抗的关键环节，但具体机制仍未明了。肉毒碱棕榈酰基转移酶1A(CPT1A)是调节脂代谢的关键酶。我们前期研究发现肝癌中脂肪酸积聚、乙酰肉碱含量减少，与肝癌恶性程度和复发转移密切相关，有诊断价值，故推测肝癌中CPT1A存在异常表达，致脂肪酸积聚引起ROS升高，参与肝癌失巢凋亡抵抗。预实验结果提示上调CPT1A后肝癌细胞内ROS显著提高，抗凋亡和克隆形成能力增强。本课题将在明确肝癌中CPT1A表达与复发、转移相关性的基础上，构建CPT1A过表达/沉默肝癌细胞系和条件性敲除小鼠模型，通过失巢模拟实验研究CPT1A过表达、ROS升高与抗失巢凋亡的关系，探究CPT1A调控ROS升高进而促进肝癌细胞入血后失巢凋亡抵抗和远处转移的发生机制，并在动物实验中予以验证，为阻断肝癌转移提供实验依据。

在我国，肝细胞癌为仅次于肺癌的第二大常见癌症且其发病机制至今仍未完全明确。目前，能量代谢在肿瘤发生机制中的作用越来越受到重视，能量代谢重编程对肿瘤发生发展机制的影响成为热点话题，但脂肪酸β氧化在肝癌中作用的相关研究相对较少。我们的研究发现CPT1A在肝癌组织中低表达，与肝内肿瘤数量及TNM分期相关，且与患者预后正相关。在低氧诱导下，HIF-1α能够负向调控CPT1A的转录。同时，CPT1A可通过抑制肝癌细胞糖酵解从而抑制肝癌发展并增加肝癌细胞对仑伐替尼的敏感性。明确了在肝癌中，CPT1A在低氧诱导下低表达并以此促进肝癌细胞糖酵解进而促进肝癌发展的这一过程，增加了肝癌发展机制中对能量代谢异常的认识。同时为仑伐替尼治疗肝癌过程中耐药的产生提供了一定的理论基础。