腺苷酸活化蛋白激酶促进MET过程对胃癌细胞增殖和侵袭力的影响及机制研究

Adenosine monophosphate activated protein kinase (AMPK) is closely related with tumor cell growth, proliferation, apoptosis and metastasis of digestive system. Our previous work found that AMPK activation of activator increased the cell junction formation of the epithelial cells and promoted the mesenchymal-epithelial transition(MET) process of such the cells;The IHC staining results was showed a membrane and luminal type with high expression patterns of AMPK in highly differentiated gastric adenocarcinoma tissues and negatively correlated with the expression of HIF-1 α.These results suggest that AMPK play a key role in regulation of MET process of the tumor cells and has a potential impact on biological behavior of tumor.For now the detailed molecular mechanism is not clear. In this study,we use the human gastric cancer cells in vitro model, use activator and inhibitor of AMPK, and establish the AMPK gene knockdown and overexpression cell model of human gastric cancer, to detect the proliferation of gastric cancer cells and the invasive ability, and further to detect the expression of AMPK/LKB1 kinase and its downstream MET related molecules, and combine with the expression of AMPK for the clinical and pathological analysis of human gastric cancer. To investigate the potential and mechanism of AMPK promoting MET process and its inhibitory effect on invasion and proliferation of gastric cancer cells.The further study provides a potential new target for clinical diagnosis and treatment of gastric cancer, and provides the experimental basis for the anti tumor treatment medicine.

最近的研究发现腺苷酸活化蛋白激酶(AMPK)与消化系肿瘤细胞的生长、增殖、凋亡及侵袭转移等过程密切相关。我们前期的工作发现AMPK激活激活剂增加上皮细胞间连接形成，促进MET过程；同时人高分化胃腺癌组织AMPK呈腔缘型膜定位高表达模式，与HIF-1α表达呈负相关，结果提示AMPK 存在对肿瘤细胞MET过程的调控并对肿瘤生物学行为产生潜在影响，目前其具体分子机制还不清楚。本课题利用人胃癌细胞的体外培养，通过AMPK激活剂及抑制剂处理，建立AMPK基因敲减及过表达人胃癌细胞模型，检测胃癌细胞的增殖和侵袭能力，进一步检测AMPK上游激酶LKB1以及其下游MET相关分子的表达，并结合人胃癌组织AMPK表达的临床病理分析工作，旨在明确AMPK促进 MET过程对胃癌细胞增殖和侵袭能力的潜在抑制性作用及其机制，为胃癌的临床诊断和治疗提供潜在的新靶点，为相关药的抗肿瘤治疗研究提供实验依据。

癌症已成为我国五大致死性疾病之首，胃癌是我国发病率仅次于肺癌的第二大恶性肿瘤，侵袭转移是导致胃癌患者致死的主要原因，而EMT是肿瘤细胞转移的起始及关键环节。AMPK是一种细胞内的能量感受器和调节器，以维持机体能量代谢平衡。肿瘤细胞可以通过代谢重组，克服因过度增殖导致的能量供应危机。细胞的能量代谢受多条信号通路的调节，其中AMPK信号通路是最重要的能量调节途径。多项研究表明，AMPK基因缺失或活性不足是恶性肿瘤发生发展的重要原因之一。本课题利用人胃癌细胞的体外培养，通过 AMPK激活剂及抑制剂处理，建立 AMPK 基因敲减及过表达人胃癌细胞模型，检测胃癌细胞的增殖和侵袭能力，进一步检测 AMPK下游EMT相关分子的表达，并结合人胃癌组织AMPK表达的临床病理分析工作，旨在明确 AMPK 促进 MET 过程对胃癌细胞增殖和侵袭能力的潜在抑制性作用及其机制。结果表明：1、二甲双胍、AICAR作用后，胃癌细胞中AMPK被磷酸化激活，呈时间和剂量依赖性；2、AMPK激活剂对胃癌细胞增殖、EMT过程和侵袭能力均有抑制作用，应用RNA干扰技术可削弱AMPK激活剂对胃癌细胞的抑制作用；3、激活AMPK后胃癌细胞中YAP蛋白发生磷酸化失活，在细胞内亚定位发生改变，由细胞核转位入胞浆，同时，YAP下游调节蛋白发生相应的改变；4、AMPK在胃癌组织中低表达，与肿瘤的分化程度、TNM分期密切相关，而与患者年龄、性别和肿瘤大小无关；YAP在胃癌组织中呈高表达，与肿瘤的大小、分化程度和TNM分期正相关，与患者年龄和性别无关。上述结果说明，AMPK通过调控YAP细胞内亚定位及磷酸化水平抑制胃癌细胞增殖、EMT和侵袭能力，为AMPK抗肿瘤机制奠定理论基础，为AMPK激活剂的抗肿瘤治疗研究提供实验依据。