miR-133b靶向EGFR通过integrin β4/FAK通路影响食管鳞癌侵袭转移的机制研究

The invasion and metastasis plays an important part in the failure of esophageal squamous cell carcinoma treatment and the intervention of its pathway is conducive to control cancer progression, which receives much concern in recent years. Through analysis of data from some related experiments, my group preliminarily discovered a possible mechanism that miR-133b -mediated EGFR may inhibit the invasion and metastasis of esophageal squamous cell carcinoma due to the suppressed expression of Grb2 and inhibited Integrin β4/FAK pathway. To confirm miR-133b effect above, over-expression and knockdown technique will be performed. In order to demonstrate the inhibition mechanism of miR-133b in esophageal squamous cell carcinoma invasion and metastasis, to begin with, we’ll determine the effects of miR-133b over-expression and knockdown on the expression of EGFR, Grb2 and Integrin β4 /FAK pathway in esophageal squamous cell carcinoma cell lines. What’s more, Dual-Luciferase Reporter Assay System will be performed to identify miR-133b effect on the direct targets to EGFR. In addition, the expression of EGFR and FAK will be reversed in miR-133b over-expression and knockdown cell lines respectively, and wound-healing, Transwell migration assay, nude mouse pulmonary xenograft model and western blot will be carried out to analyze the role of Integrin β4 /FAK pathway and EGFR in inhibiting of invasion and metastasis of esophageal squamous cell carcinoma due to miR-133b. Furthermore, chromatin immunoprecipitation will be employed to analyze the Grb2 protein effect on Integrin β4 promoter sequence to confirm whether Grb2 molecular could directly regulate Integrin β4 expression or not. Last but not the least, in-situ hybridization, immunohistochemistry and statistics will be used to investigate the association between the expression levels of miR-133b, EGFR, Grb2, Integrinβ4, FAK in esophageal squamous cell carcinoma tissues, and analyze the relationship between the expression levels of these molecular and clinical stage, tumor size, metastasis, infiltration depth and survival rates of esophageal squamous cell carcinoma patients. The project will reveal the innovative mechanism of miR-133b in inhibiting invasion and metastasis of esophageal squamous cell carcinoma by inhibiting EGFR expression, regulating Grb2 expression, suppressing ntegrin β4 /FAK Pathwy and lay a newly theoretic foundation for the identification of novel potential therapeutic targets for treating esophageal squamous cell carcinoma patients.

侵袭、转移是食管鳞癌治疗失败的主因，干预侵袭转移途径，利于控制肿瘤的进展。本项目前期实验发现：miR-133b可能靶向EGFR，调控Grb2表达，通过integrin β4/FAK通路，抑制食管鳞癌的侵袭转移。为证实此功能，采用过表达和沉默技术，在体内外实验研究miR-133b对食管鳞癌侵袭转移的影响。为明确其机制，采用双荧光素酶报告基因系统验证miR-133b对EGFR的靶向功能；通过逆转EGFR及FAK表达，验证EGFR、integrin β4/FAK是否为miR-133b抑制食管鳞癌侵袭转移的直接靶点和关键信号通路；应用ChIP实验验证Grb2对integrin β4的调控作用；在食管鳞癌组织中验证上述分子的表达特征及与生存的关联，从而阐明miR-133b-EGFR-Grb2-integrin β4/FAK轴调控食管鳞癌侵袭转移的机制，为食管鳞癌的治疗新靶点提供理论基础。

食管鳞癌是中国常见的恶性肿瘤之一，位居恶性肿瘤引发死亡的前列。miR-133b在食管鳞癌组织中呈低表达，miR-133b 的表达与肿瘤的淋巴结转移、 浸润深度呈负相关，提示miR-133b作为抑癌基因参与食管鳞癌的发生、发展过程。基于上述背景，本项目从以下三个方面进行研究：（1）采用体内外实验鉴定miR-133b抑制食管鳞癌侵袭、转移的生物学功能。（2）在过表达及沉默表达基础上，利用细胞内分子逆转技术， 从细胞水平验证miR-133b是否通过直接靶向EGFR，通过integrinβ4/FAK信号通路影响食管鳞癌细胞的侵袭、转移。（3）在人食管鳞癌组织中验证miR-133b-EGFR-ITGB4/FAK轴与食管鳞癌侵袭、转移的调控关系。 通过上述研究将阐明miR-133b通过直接靶向EGFR，抑制ITGB4/FAK通路的活化，从而影响食管鳞癌侵袭、转移的分子机制。研究结果显示，食管鳞癌组织和细胞中miR‑133b水平降低，且与EGFR、ITGB4、FAK的表达呈负相关（P＜0.05，P＜0.01，P＜0.01）。miR‑133b可以下调EGFR的表达，通过靶向ITGB4/FAK通路抑制食管鳞癌细胞增殖、抵抗失巢凋亡、迁移、侵袭和上皮间充质转化的特性（P＜0.05，P＜0.01，P＜0.05，P＜0.01，P＜0.01）。通过上述研究将为食管鳞癌的分子靶向治疗向临床转化打下理论基础，为调控食管鳞癌侵袭、转移机制的研究提供新的线索。