STMN1通过调控p38MAPK/STAT1信号通路介导肺腺癌转移的作用及机制研究

Metastasis is the leading cause of death in patients with lung adenocarcinoma, which has no available treatment. Our previous study found that over-expression of STMN1 in lung adenocarcinoma tissue showed to be closely correlated with the activation of p38 MAPK and STAT1, as well as lymphatic metastasis; activation of p38 MAPK and STAT1 can be inhibited by down-regulating STMN1 expression, metastatic potential be decreased significantly also. Therefore, we hypothesize that "STMN1 may promote EMT through p38 MAPK/STAT1 pathway in lung adenocarcinoma". Firstly, STMN1 will be overexpressed or knockouted by gene transfection or CRISPR/Cas9 in lung adenocarcinoma cell lines, which would be used to explore its function in modulating p38 MAPK/STAT1 pathway and EMT formation. Secondly, the mechanism, STMN1 controlling p38 MAPK/STAT1 pathway to regulate EMT via modulation of MT stability, will be explicited by using site-specific mutagenesis, RNAi, fluorescence resonance energy transfer. Thirdly, the effect and mechanism of STMN1 modulating EMT via p38 MAPK/STAT1 pathway will be valided by means of IHC and so on. Our research will not only provid new evidences for the treatment of advanced lung adenocarcinoma, but also have wide application prospects.

转移是肺腺癌患者死亡的主要原因，但迄今尚无有效的治疗方法。我们前期发现肺腺癌组织中STMN1表达与p38 MAPK及STAT1活化、肺腺癌淋巴转移密切相关；下调其表达可抑制p38 MAPK及STAT1活化并抑制肺腺癌转移。故提出“STMN1通过调控p38 MAPK/STAT1信号通路促进肺腺癌EMT发生”的假设。本课题应用基因转染和CRISPR/Cas9技术构建STMN1上调及敲除细胞模型，研究STMN1在调节p38 MAPK/STAT1通路活化及肺腺癌EMT中的作用；应用定点突变、RNAi、荧光能量共振转移等方法明确STMN1通过p38 MAPK/STAT1通路调控微管稳定性介导肺腺癌EMT发生的机制；应用免疫组化等方法在裸鼠模型及临床标本中验证STMN1通过p38 MAPK/STAT1通路介导肺腺癌EMT发生的作用及其机制。该研究将为靶向治疗肺腺癌转移提供新的依据，具有广阔的应用前景。

肺腺癌是目前发病率最高的肺癌亚型，由于缺乏有效的早期诊断措施及其易发生转移的特性，肺腺癌的五年生存率仍较低。在前期研究中我们发现，STMN1在肺腺癌组织中高表达，且其高表达与肺腺癌淋巴结转移、p38 MAPK 、STAT1 活化呈正相关。因此，我们推测STMN1通过p38MAPK/STAT1信号通路介导肺腺癌EMT的发生。在本研究中，我们通过CRISPR/CAS9及慢病毒转染技术成功构建STMN1敲减及过表达细胞株。研究发现，敲减STMN1后肺腺癌细胞的迁移能力受到明显抑制，且p38及STAT1的活化水平显著降低，而过表达STMN1后肺腺癌细胞的迁移能力明显增强，p-p38及p-STAT1表达水平显著上调。为了进一步验证该假设，我们用p38MAPK通路抑制剂SB203580及STAT1抑制剂氟拉达滨对肺腺癌细胞进行处理。结果表明，抑制p38MAPK/STAT1通路可显著逆转因过表达STMN1引起的EMT增强。同时，我们发现STAT1可能与ZEB1的启动子区域结合。抑制STAT1的表达可导致ZEB1表达显著降低。STMN1是微管解聚蛋白，我们通过紫杉醇及秋水仙碱处理细胞后观察微管稳定性与EMT的关系。我们发现紫杉醇可以抑制过表达STMN1引起的EMT增强；秋水仙碱对EMT的影响则与过表达STMN1一致。这表明STMN1可通过调节微管稳定性介导EMT的发生。为进一步探索STMN1介导肺腺癌 EMT发生的机制，我们通过质谱鉴定发现STMN1与 HMGA1存在互相作用。HMGA1已被证实可促进EMT的发生，但HMGA1属于构架性转录因子，其无直接的转录激活能力，STMN1与HMGA1结合，可激活HMGA1的转录调节活性。同时，我们发现HMGA1与微管相关基因显著共表达，故推测HMGA1可能与 STMN1存在协同作用，通过影响微管稳定性介导肺腺癌EMT的发生。微管稳定性，HMGA1及STMN1之间的关系仍需进一步探索。综上所述，我们发现STMN1不仅可以通过p38MAPK/STAT1信号通路介导肺腺癌EMT的发生，同时也可以通过调节微管稳定性介导EMT的发生；另外， STMN1可与 HMGA1相互作用，促进EMT的发生。我们的研究结果阐明了STMN1介导肺腺EMT发生的部分机制，这有助于我们更好地认识肺腺癌的转移机制，为靶向治疗肺腺癌提供依据，具有广阔的应用前景。