Wang Nina, Xie Jian-Ming, Zheng Da-Yong, Zuo Qiang, Liao Wang-Jun
Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2012 Mar;32(3):312-6.
To establish a gastric cancer cell line with stable expression of metastasis-associated in colon cancer 1 (MACC1) and detect the changes in tumor-related gene expression profiles for investigating the possible regulation mechanisms between MACC1 and the differentially expressed genes.
The full-length MACC1 cDNA was amplified from human embryonic kidney 293FT cells and cloned into the pBaBb-puro vector. The recombinant pBaBb-puro-MACC1 expression vector, after identification with restriction enzyme digestion, was transfected into 293FT cells, and the expression of fluorescent reporter gene was observed. pBaBb-puro-MACC1 vector was transfected into human gastric cancer BGC-823 cell line to establish BGC-823/pBaBb-puro-MACC1 cell line stably expressing MACC1. Quantitative RT-PCR and Western blotting were used to detect MACC1 expression in both BGC-823/pBaBb-puro-MACC1 and control BGC-823 cells. High-throughout cDNA microarray was used to screen the effects of MACC1 on the gene expression profiles of gastric cancer cells.
The recombinant pBaBb-puro-MACC1 plasmid was successfully constructed and verified by PCR and sequencing. BGC-823/pBaBb-puro-MACC1 cells showed significantly increased MACC1 mRNA expression as compared with the control cells. The results of cDNA microarray identified 33 up-regulated and 24 down-regulated genes in the cells after MACC1 transfection involved were in various cellular functions.
The established BGC-823/pBaBb-puro-MACC1 gastric cancer cell line show some important molecular changes caused by MACC1.
建立稳定表达结肠癌转移相关蛋白1（MACC1）的胃癌细胞系，并检测肿瘤相关基因表达谱的变化，以探讨MACC1与差异表达基因之间可能的调控机制。
从人胚肾293FT细胞中扩增出MACC1 cDNA全长，克隆至pBaBb-puro载体。经限制性内切酶酶切鉴定后的重组pBaBb-puro-MACC1表达载体转染至293FT细胞，观察荧光报告基因的表达情况。将pBaBb-puro-MACC1载体转染至人胃癌BGC-823细胞系，建立稳定表达MACC1的BGC-823/pBaBb-puro-MACC1细胞系。采用定量逆转录-聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测BGC-823/pBaBb-puro-MACC1细胞及对照BGC-823细胞中MACC1的表达。利用高通量cDNA微阵列筛选MACC1对胃癌细胞基因表达谱的影响。
成功构建重组pBaBb-puro-MACC1质粒，并经PCR和测序验证。与对照细胞相比，BGC-823/pBaBb-puro-MACC1细胞中MACC1 mRNA表达显著增加。cDNA微阵列结果显示，MACC1转染后的细胞中有33个基因上调，24个基因下调，这些基因涉及多种细胞功能。
所建立的BGC-823/pBaBb-puro-MACC1胃癌细胞系显示出由MACC1引起的一些重要分子变化。
