稳定表达MACC1的胃癌BGC-823/pBaBb-puro-MACC1细胞株的建立及其肿瘤相关基因的研究基因

Establishment of BGC-823/pBaBb-puro-MACC1 gastric cancer cell line stably expressing MACC1 and its tumor-related gene expression profiles

目的构建含有全长肠癌转移相关基因(MACC1)的表达载体,建立稳定表达MACC1的胃癌BGC-823/pBaBb-puro-MACC1细胞系,通过基因芯片技术筛选出转染MACC1后胃癌细胞株的差异表达基因,探讨MACC1基因与差异基因之间可能的调节机制或信号通路。方法以人胚肾293FT细胞为模板,经PCR扩增获全长MACC1 cDNA序列,将目的基因序列克隆入带有绿色荧光蛋白报告基因的pBaBb-puro表达载体,建立pBaBb-puro-MACC1表达载体。经限制性内切酶酶切鉴定、测序并转染293FT细胞观察报告基因表达情况判断是否构建成功。构建成功后的pBaBb-puro-MACC1表达载体转染人胃癌BGC-823细胞,建立稳定表达MACC1的BGC-823/pBaBb-puro-MACC1细胞。qRT-PCR及western blot检测转染细胞的MACC1基因表达,应用基因芯片技术筛选出转染MACC1基因后胃癌细胞株的差异表达基因并对其探讨研究。结果成功扩增全长MACC1cDNA,经测序鉴定序列完全正确;转染293FT细胞可见清晰的绿色荧光表达。BGC-823/pBaBb-puro-MACC1细胞能够稳定表达MACC1。基因芯片筛选出差异基因发现上调基因33个,下调基因24个,这些差异表达基因的功能涉及多个方面。结论成功构建了含有全长MACC1cDNA的表达载体系统,建立了稳定表达MACC1的BGC-823/pBaBb-puro-MACC1细胞,通过对差异基因的分析发现MACC1可能引起胃癌细胞株BGC-823基因表达谱中一些非常重要的分子的改变,为MACC1基因进一步研究奠定了良好基础。

Objective 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.Methods 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.Results 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.Conclusion The established BGC-823/pBaBb-puro-MACC1 gastric cancer cell line show some important molecular changes caused by MACC1.