能量代谢相关酶ALDOA在肺癌EMT中的作用及其机制研究

The role of glucose metabolism abnormity in tumorigenesis has drawn more and more attention.And the relationship between glycolytic enzymes related with glycometabolism and tumorignensis has became a focus. Seeking glycolytic enzyme inhibitors to suppress the genesis and development of lung cancer effectively has become one of the ideal strategies for the cancer therapy. We examined tumor and adjacent normal tissues to identify differential expressed proteins by a DIGE approach combined with mass spectrometry. Fructose-1,6-bisphosphate aldolase A (ALDOA) was one of differential expressed proteins, and our preliminary studies indicated that ALDOA played an important role in the genesis and metastasis of lung cancer.We plan to go in for further studies, including investigating the effects of ALDOA on ablities of cell cycle , cell proliferation, invasion , metastasis and EMT of lung cancer in vitro and in vivo by using DIGE, mass spectrometry, transfection, Real-time PCR and gene knockout methods. And we attempt to elucidate the influence and the molecular mechanism of how ALDOA participates in the tumor genesis and progression. Meanwhile, as a secretory protein produced by tumor abnormal metabolism, ALDOA can be detected in the peripheral circulation. Appraising the feasibility of clinical diagnosis for ALDOA is of great significance in making non-invasive or minimally invasive cancer diagnosis.

糖代谢异常在肿瘤发生中的作用越来越引起人们的关注，与糖代谢相关的糖酵解酶与肿瘤的关系成为关注的焦点。寻求糖酵解酶抑制剂，有效地抑制肿瘤的发生发展成为肿瘤治疗的新策略。果糖二磷酸醛缩酶A(ALDOA)是我们在临床样本中用DIGE结合质谱技术筛选出的肿瘤组织与癌旁组织差异蛋白，前期研究提示ALDOA在肺癌的发生和转移中均有重要作用。本课题利用DIGE、质谱、转染、Real-time PCR、基因敲除等手段，通过体内外实验探讨ALDOA对肺癌细胞周期、细胞增殖、EMT、侵袭转移方面的影响，明确ALDOA对肿瘤发生发展的影响和分子机制。同时，ALDOA作为肿瘤异常代谢中产生的分泌蛋白，可以实现外周血的有效检测，评估ALDOA的临床诊断意义对于实现微创或无创肿瘤诊断具有重要意义。

与糖代谢相关的糖酵解酶与肿瘤的关系已成为研究焦点。寻求糖酵解酶抑制剂，有效地抑制肿瘤的发生发展成为肿瘤治疗的新策略。本课题完成了以下几方面的工作：（1）果糖二磷酸醛缩酶A(ALDOA)是我们在临床样本中应用2D-DIGE结合MALDI-TOF/TOF质谱鉴定技术筛选出的差异蛋白。ALDOA在肺癌组织中高表达。利用Western Blot在17对肺鳞癌组织及其配对的癌旁肺组织以及肺鳞癌组织芯片中对ALDOA进行了验证， 其结果与蛋白质组学结果的差异趋势一致；（2）经分析ALDOA的表达情况和75例肺鳞癌患者临床病例资料中的各项指征，发现ALDOA的表达与肺鳞癌病理分期呈正相关趋势，ALDOA的表达与肿瘤转移呈正相关，ALDOA的高表达与患者术后无瘤生存时间呈负相关，ALDOA的表达与性别、年龄、肿瘤部位和分化程度均无相关性；对29例肺癌样本中ALDOA的表达与分期的相关性进行分析，发现其表达与分期无相关性；对27例肺癌样本中ALDOA的表达与患者淋巴结转移的相关性进行分析，发现其表达与淋巴结转移无相关性、与患者的性别、年龄和肺癌病理类型均无相关性；（3）通过稳转建立了ALDOA低表达稳定克隆细胞系，发现ALDOA表达下调抑制NCI-H520细胞体外生长、迁移、侵袭和致瘤能力，促进细胞凋亡；并使细胞G1/S期和G2/M期受到阻滞；可调控增殖、EMT标记物等相关蛋白表达（促进p53、 p21 、E-cadherin，β-catenin蛋白上调，诱导Akt、Cyclin D1、PCNA、 Fibronectin、Vimentin蛋白下调）；Real-time PCR检测细胞EMT发生相关基因mRNA的表达发现，ALDOA可下调Slug、Snail、Twist和Zeb1基因，上调FOXC1基因；ALDOA表达下调抑制人肺鳞癌NCI-H520细胞体外增殖能力；（4）稳转高表达ALDOA细胞系发现其可促进NCI-H520增殖、促进NCI-H520 G1/S期阻滞、调控CyclinD1/CDK4-Rb通路、可增加NCI-H520中糖代谢限速酶PKM2蛋白的含量并促进其核定位、通过MEK/ERK通路影响NCI-H520的细胞增殖和Cyclin D1表达。以上研究结果为临床转移预警提供基础资料。在此期间，共发表SCI收录的论文11篇，其中标注的6篇。