利用cDNA微阵列分析EMP-1在食管癌细胞中诱导的基因表达谱。
Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray.
作者信息
Wang Hai-Tao, Kong Jian-Ping, Ding Fang, Wang Xiu-Qin, Wang Ming-Rong, Liu Lian-Xin, Wu Min, Liu Zhi-Hua
机构信息
National Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100021, China.
出版信息
World J Gastroenterol. 2003 Mar;9(3):392-8. doi: 10.3748/wjg.v9.i3.392.
AIM
To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1.
METHODS
The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component.
RESULTS
Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion.
CONCLUSION
Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.
目的
构建携带人上皮膜蛋白-1（EMP-1）开放阅读框（ORF）的真核表达质粒，转染人食管癌EC9706细胞，获得稳定表达EMP-1的细胞株，探讨EMP-1影响细胞增殖及基因谱改变的机制。
方法
构建含EMP-1 ORF的pcDNA3.1/myc-his表达载体，转染人食管癌EC9706细胞，经Western blot和RT-PCR筛选阳性克隆；绘制细胞生长曲线，用流式细胞术检测细胞周期；采用cDNA芯片技术比较阳性克隆与对照细胞基因表达谱差异，随机选取4个差异表达基因进行半定量RT-PCR验证，并根据基因功能和细胞定位进行分类。
结果
人EMP-1基因可在转染细胞中稳定表达；转染阳性克隆细胞生长速度减慢，S期阻滞，G1期延长；cDNA芯片分析显示，转染后35个基因表达水平变化2倍以上，其中28个基因上调，7个基因下调；分类基因中，近半数上调基因（28个中的13个）与细胞信号转导、细胞通讯尤其是细胞黏附相关。
结论
人EMP-1基因过表达可抑制EC9706细胞增殖，使细胞阻滞于S期，G1期延长；cDNA芯片分析提示，EMP-1可能是细胞信号转导、细胞通讯及黏附相关的调节分子之一。
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