C-myc/miR-144/COX-2通路在食管鳞癌转移中的作用

China is a country with the highest incidence and mortality of esophageal squamous cell carcinoma (ESCC). Previour studies showed COX-2 plays an important role in the development and progression of ESCC. Our recent work found that COX-2 was closely related to the metastasis of ESCC. MiRNAs have important effect on the metastasis of carcinoam. However, miRNAs, which can regulate COX-2 in ESCC, has not been reported previously. Bioinformatic analysis showed that miR-144 could bind to 3' UTR of COX-2; our preliminary studies found that expression of miR-144 was down-regulated in ESCC tissues and cells; overexpression of miR-144 inhibited the metastasis of ESCC cells and led to increased expression of COX-2. In this study, we planned to investigate whether miR-144 could bind to the 3' UTR of COX-2 and inhibite COX-2 expression by useing luciferase reporter gene. It has been reported that c-myc could affect the tumorigenesis and developing through regulating transcription of miRNAs. In this project, we planned to further study wether c-myc could regulate the transcription of miR-144 in ESCC using ChIP and luciferase reporter gene. This study will provide important data for reveal the mechanism of c-myc/miR-144/COX-2 pathway in ESCC.

我国是食管鳞癌发病率和死亡率最高的国家。研究显示COX-2在食管鳞癌发生发展中起重要作用，我们近期的研究发现COX-2与食管鳞癌的转移密切相关。MiRNAs在肿瘤的转移中起重要的调节作用。在食管鳞癌中哪些miRNAs能调节COX-2表达还未见报导。我们通过生物信息学预测发现miR-144可以和COX-2的3'UTR区结合；初步的研究结果表明在食管鳞癌组织和细胞中miR-144表达降低；提高其表达水平能抑制ESCC的转移，而且引起COX-2表达下降。在此基础上，本研究拟利用报告基因实验进一步证实miR-144 通过与COX-2 3'UTR区直接作用抑制其表达。已有的研究表明c-myc可以调节miRNAs表达影响肿瘤发生发展。本研究计划利用ChIP和报告基因实验研究是否c-myc调节了食管鳞癌中miR-144的表达。以此揭示c-myc/miR-144/COX-2调节通路在食管鳞癌转移中的作用。

研究显示COX-2和miRNAs在食管鳞癌的发生发展中起重要作用，但是在食管鳞癌中调节COX-2表达的miRNA目前尚无报导。数据库显示miR-144可以和COX-2的3’非编码区结合，在本研究中我们应用报告基因检测的方法证实了这一预测结果；同时应用实时定量PCR方法检测了30对食管鳞癌/癌旁组织和11株食管鳞癌细胞系中的表达，结果显示miR-144在食管鳞癌和癌旁组织中表达下调；进一步建立食管鳞癌miR-144过表达克隆，应用western-blot和ELISA方法检测发现过表达克隆中COX-2和其衍生物PGE2表达下降；应用CCK8检测过表达克隆增殖能力发现miR-144对食管鳞癌细胞增殖无明显抑制作用；应用transwell小室方法检测发现过表达miR-144后食管鳞癌细胞的迁移和侵袭能力明显下降；给裸鼠尾静脉注射一定数量的食管鳞癌miR-144过表达克隆，3月后发现与对照组相比裸鼠肝脏细胞表面转移灶的数量明显减少。结论：miR-144在食管鳞癌组织中表达下调，与COX-2表达呈负相关；miR-144对食管鳞癌细胞增殖无抑制作用；可以在体内和体外抑制食管鳞癌细胞的转移，这一作用可能部分通过COX-2途径完成。