MiR-214下调CrkL抑制食管鳞癌上皮-间质转化及转移的机制研究

Increasing evidence supports a role for epithelial-mesenchymal transitions (EMT) in the acquisition of migratory and invasive properties that promote the dissemination of tumor cells and metastasis. Recent reports further demonstrated that microRNA-214 (miR-214) regulate EMT in several cancers. However, the role of miR-214 in the regulation of EMT in esophageal squamous cell carcinoma (ESCC) remains unknown. In a recent work we demonstrated that the expresssion of miR-214 in ESCC tissuses was significantly lower than that in matched normal tissues,and the the expresssion of miR-214 was negatively correlated with metastasis in ESCC patients.In addition, we found that miR-214 significantly repressed the expression of CT10 regulator of kinase like protein (CrkL), which reportedly induces EMT in variety types of tumors.Moreover, over-expression of miR-214 could repress EMT in ESCC cells. We, therefore, hypothesized that miR-214 might inhibit ESCC EMT and metastasis via miR-214 mediated down-regulation of CrkL expression. In this project, we plan to evaluate the role of miR-214/CrkL in the EMT of ESCC by multiple approaches including bioinformatic analysis, luciferase reporter assay,yeast two-hybrid, ChIP, fluorescence resonance energy transfer, adenovirus- (or lentivirus-) mediated gene delivery, metastasis assay in NOD/SCID mice and clinical investigation. Conceivably, elucidation of the molecular pathways involved in the EMT of ESCC by miR-214 will provide important clues for the development and evaluation of novel anticancer therapies.

上皮-间质转化（EMT）与上皮类肿瘤的转移发生密切相关。近年来有关miRNA调控肿瘤EMT的机制研究取得了一定进展，但目前关于miRNA对食管鳞癌EMT的调控作用及其机制尚不明确。前期我们发现，食管鳞癌中miR-214的表达水平与EMT及侵袭转移呈显著负相关，细胞学实验提示miR-214可能通过下调Crk样蛋白（CrkL）表达抑制食管鳞癌EMT，但其具体的机制仍有待于进一步明确。本项目拟通过荧光素酶报告基因实验明确miR-214抑制CrkL的作用，采用酵母双杂交、生物信息学分析、免疫共沉淀、荧光共振能量转移及细胞功能实验明确CrkL调控EMT的途径，籍此建立miR-214下调CrkL表达抑制食管鳞癌EMT的信号通路；此外，通过动物转移成瘤模型及临床统计分析评价miR-214、CrkL及CrkL靶基因在食管鳞癌转移诊疗中的潜在应用价值，为今后临床开展食管鳞癌转移相关的诊疗新方法提供理论依据。

食管鳞癌(ESCC)是严重威胁人类健康和生存的恶性肿瘤疾病。前期研究结果显示，miR-214可以通过抑制食管鳞癌EMT进而抑制其侵袭和转移，但其在食管鳞癌中的具体调控机制尚不明确。.在本项目中，我们通过染色质免疫共沉淀、荧光素酶报告基因及相关细胞功能实验明确在食管鳞癌细胞中miR-214对CrkL表达的调控作用及具体机制；进而通过细胞功能实验探讨miR-214-CrkL途径中关键基因对食管鳞癌细胞迁移、侵袭及EMT的调控作用；此外我们还通过免疫缺陷小鼠成瘤实验和临床调查对食管鳞癌中miR-214-NANOG途径中关键基因表达水平与肿瘤侵袭转移影响的相关性作进一步的探讨。 .研究结果显示，食管鳞癌细胞中miR-214可直接抑制CrkL的表达，进而抑制食管鳞癌细胞迁移侵袭和EMT。随后我们通过CoIP对CrkL下游潜在靶基因进行筛选，并加以验证。结果显示PTK2蛋白与CrkL相互结合，表达检测结果显示CrkL不影响PTK2表达，但影响其磷酸化进而增强其活性。细胞功能实验结果显示，CrkL及PTK2过表达具有促进食管鳞癌细胞EMT及侵袭迁移的作用，动物成瘤实验结果显示，过表达CrkL及PTK2可显著增强食管鳞癌细胞体内致瘤能力。临床病理分析结果显示，miR-214-CrkL途径中关键基因表达水平与与肿瘤的分期、分化程度及转移相关，而与患者年龄、性别等无显著相关性。.我们的研究发现miR-214可以通过抑制CrkL表达进而影响PTK2磷酸化活化，进而抑制食管鳞癌细胞的EMT、迁移及侵袭能力。通过动物实验及临床调查证实miR-214-CrkL途径与食管鳞癌进展相关。