食管癌细胞ezrin基因启动子和增强子的靶向敲除及生物学功能研究

Ezrin gene is overexpressed significantly in esophageal squamous cell carcinoma. There is a relationship between overexpression of ezrin and metastatic potential of carcinoma cells, but the molecular mechanism underlying its regulated expression remain unclear. Recently, we identified the regions of ezrin promoter and enhancer. Results of reporter gene assay revealed that ezrin enhancer possessed dual function: transcriptional enhancement and activation. These suggest that the overexpression of ezrin in carcinoma cells should be probably associated with the special transcriptional regulatory properties of ezrin enhancer. Based on these results, we investigate the biological function of ezrin promoter and enhancer in the present study. We will firstly construct a series of targeting vectors for ezrin promoter and enhancer using DNA clone techniques, then knockout ezrin promoter and enhancer in human esophageal carcinoma cells using homologous recombination techniques, further detect the effect of the target sequences knockout on ezrin expression and biological behavior of esophageal carcinoma cells using experimental techniques of gene expression assay, cell metastasis and xenografts in nude mice, and finaly elucidate the biological function of ezrin gene promoter and enhancer in human esophageal carcinoma cells. The expected findings would not only benefit to further research on the regulatory mechanisms of ezrin overexpression, but also provide a new way to explore molecular targets for assistant diagnosis and biotherapy against carcinoma.

ezrin基因在食管癌细胞中显著过表达，与癌细胞的侵袭转移相关，但其过表达调控机制不明。最近，我们鉴定出ezrin增强子和启动子区，报告基因检测结果显示，增强子区同时具有转录增强和转录起始双重功能，提示肿瘤细胞中ezrin基因过表达可能与其增强子区独特的转录调控活性有关。本研究将在既往工作基础上，根据上述最新研究进展，进行ezrin启动子和增强子生物学功能研究。本研究采用DNA克隆技术构建ezrin启动子和增强子打靶载体；采用同源重组技术靶向敲除食管癌细胞株ezrin启动子和增强子；采用基因表达分析、细胞侵袭移动和裸鼠移植瘤等实验技术，检测目标序列的敲除对食管癌细胞的ezrin基因表达和肿瘤生物学行为的影响；最终明确ezrin启动子和增强子在食管癌细胞中的生物学功能。本研究预期结果不仅为后续系统的ezrin基因过表达调控机制研究奠定基础，也将为癌症的辅助诊断、判断预后和治疗研究提供新途径。

ezrin基因在食管癌细胞中显著过表达，与癌细胞的侵袭转移相关，但其过表达调控机制不明。最近，我们鉴定出ezrin增强子区，报告基因检测结果显示，增强子区同时具有转录增强和转录起始双重功能，提示肿瘤细胞中ezrin基因过表达可能与其增强子区独特的转录调控活性有关。本研究在既往工作基础上，根据上述最新研究进展，进行ezrin增强子生物学功能研究。首先利用生物信息学分析技术，设计筛选一系列靶向ezrin增强子的gRNA，构建CRISPR-Cas9基因编辑重组表达载体，验证gRNA的靶向活性。然后将重组质粒共转染食管癌细胞，经嘌呤霉素筛选及DNA测序鉴定，获得依靠基因编辑技术靶向敲除ezrin增强子的单克隆食管癌细胞株。对目的基因进行实时定量PCR和Western blot检测，结果显示，靶向敲除ezrin增强子降低食管癌细胞ezrin基因的mRNA和蛋白质的表达水平。细胞增殖实验和裸鼠移植瘤实验结果显示，ezrin增强子敲除肿瘤细胞的细胞增殖速率和致瘤性有所降低。结果表明，ezrin增强子具有调控食管癌细胞ezrin基因表达的作用，敲除ezirn增强子能够降低基因的表达，进而降低食管癌细胞增殖和裸鼠致瘤。本研究结果为后续系统研究ezrin基因过表达调控机制奠定基础。ezirn增强子有望作为一个有效靶点，为癌症的辅助诊断、判断预后和治疗研究提供新途径。