AMPK经ZO-1抑制人舌癌细胞增殖和迁移的分子机制

As a sensor of cellular energy status in all eukaryotic cells, AMP-activated protein kinase (AMPK) has been implicated to play a critical role in the regulation of cell proliferation, apoptosis and migration in various cancers. Disruption of normal structure and loss of function of tight junction involve in the carcinogenesis and tumor metastasis. However, whether this process is mediated by AMPK as well as its underlying mechanisms are unclear. In our previous work, we found that the expression of ZO-1 protein was significantly decreased in tumor tissues of tongue compared with paracancer. After activation of AMPK in human tongue squamous cell carcinoma cell line SCC15, ZO-1 was translocated to membrane and the proliferation and migration of SCC15 cells were inhibited, suggesting that AMPK might inhibit the proliferation and migration of human tongue squamous cell carcinoma cells via ZO-1. The objectives of this proposal are: 1) to confirm whether the inhibitory effect of AMPK on proliferation and migration of SCC15 cells is mediated by dysfunction of tight junction by measurement of Transepithelial Electrical Resistance (TER); 2) to examine the distribution and phosphorylation of ZO-1 after activation of AMPK and to clarify the effect of ZO-1 on cell proliferation and migration by gene knock-down or overexpression and determine the regulatory role of AMPK in this process. 3) to explore the downstream molecules in AMPK/ZO-1 signaling pathway, cyclins and cyclin-dependent kinases involved in proliferation, and microfilament-associated proteins involved in migration will be investigated. This study will provide us with useful information to better understand the molecular mechanisms of AMPK inhibiting the proliferation and migration of human tongue squamous cell carcinoma cells and to further explore new molecular targeted therapy for tongue squamous cell carcinoma.

AMPK作为细胞代谢调节器可调节细胞增殖、凋亡和迁移。紧密连接结构的破坏和功能的丧失参与肿瘤的发生及转移，但该过程是否受AMPK调控及其机制尚不清楚。我们前期发现舌癌组织中ZO-1表达降低，在舌癌细胞SCC15中激活AMPK后，ZO-1向胞膜转位，SCC15增殖和迁移被抑制，推测AMPK可经ZO-1抑制舌癌细胞的增殖和迁移。我们拟①测定跨上皮细胞电阻（TER）等以证实AMPK通过破坏紧密连接抑制舌癌细胞的增殖和迁移；②激活AMPK后观察ZO-1的分布与磷酸化变化；敲低或过表达ZO-1以明确其对舌癌细胞增殖和迁移的作用，并证明该过程受AMPK调节；③以与细胞增殖相关的细胞周期蛋白及其激酶和与迁移相关的微丝结合蛋白为靶分子，确定AMPK/ZO-1通路的下游事件。该研究将有助于揭示激活AMPK抑制舌癌细胞增殖和迁移的分子机制，并可为临床上以AMPK为靶点进行舌癌的分子靶向治疗提供理论依据。

本研究探讨腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）对舌癌细胞增殖和迁移的影响，及其对舌癌细胞紧密连接结构和功能的作用，证实AMPK经紧密连接分子ZO-1抑制舌癌细胞的增殖和迁移，并阐明其相关的作用机制。研究结果表明：①用AICAR（AMPK的激动剂）作用于不同的人舌癌细胞，均能激活各舌癌细胞AMPK的激酶活性， 且可使细胞周期发生阻滞，显著抑制各舌癌细胞的增殖和迁移，提示激活AMPK是抑制舌癌细胞增殖和迁移的重要原因；②Western blot和免疫组化方法检测37例舌癌标本，发现舌癌组织紧密连接分子ZO-1表达显著下调，p-AMPK水平在舌癌早期及高分化舌癌组织中则显著上调。用AICAR（1mM）激活AMPK可特异性上调周期调节蛋白p21、p27及p53的表达，显著上调舌癌细胞ZO-1蛋白的表达，使ZO-1分子发生膜转位，并降低舌癌细胞的跨上皮细胞电阻（TER），增加其对4 kDa FITC-右旋糖酐的旁细胞通透性，同时促进细胞骨架F-actin环带微丝的断裂，提示周期蛋白调节因子表达的改变、紧密连接结构和功能的破坏、及细胞骨架的解聚是AMPK抑制舌癌细胞增殖或迁移可能的机制；③用siRNA技术敲低ZO-1的表达后激活AMPK，发现激活AMPK对细胞增殖和迁移的抑制作用被部分逆转，提示AMPK对舌癌细胞增殖和迁移的抑制作用部分依赖于ZO-1；④ZONAB是ZO-1相关的转录因子，ZO-1 可以结合 ZONAB 并通过抑制其入核进而抑制其活性，调节细胞增殖。我们发现激活AMPK可使ZONAB表达上调，敲低ZO-1可特异性地抑制ZONAB的表达；丝切蛋白Cofilin是一种可以促进F-actin微丝解聚的微丝结合蛋白，调节细胞的迁移。我们发现激活AMPK可上调Cofilin的表达，敲低ZO-1可特异性地抑制Cofilin的表达。以上结果提示，ZONAB和Cofilin可能是AMPK经ZO-1抑制舌癌细胞增殖和迁移的重要下游分子。本研究证实了激活AMPK可抑制舌癌细胞的增殖和迁移，该作用可能是部分通过调节ZO-1分子的表达和分布，进而调节ZONAB和Cofilin 的表达而实现的。本研究为揭示AMPK调控舌癌细胞增殖和迁移的分子机制、探讨紧密连接与肿瘤增殖和迁移的关系提供了实验依据，为临床上以AMPK为靶点进行舌癌的分子靶