表达谱测序筛选大肠癌colo205细胞及其来源的CD133^+、CD133^-细胞群肿瘤代谢相关基因

Gene expression profiling for screening of cancer metabolism related genes in CD133^+/CD133^- colorectal cancer cells derived from colo205 cells

目的:获得colo205细胞株不同细胞群肿瘤代谢基因及代谢调控基因的差异表达变化信息,为深入理解大肠癌肿瘤的发生机制、药物治疗潜在靶点的发现及大肠癌的临床治疗提供实验依据.方法:利用无血清体外培养、磁珠分选colo205细胞株来源的大肠癌CD133+细胞群、CD133-细胞群结合裸鼠成瘤实验验证后,表达谱测序结合生物信息学分析筛选不同细胞群差异表达肿瘤代谢基因及代谢调控基因,并用定量PCR技术进行验证.结果:与CD133-细胞群比较,CD133+细胞群有9个癌症代谢基因表达发生上调,3个癌症代谢基因表达发生下调,colo205未分选细胞群有12个癌症代谢基因表达发生上调,3个癌症代谢基因表达发生下调.与CD133+细胞群比较,colo205细胞有5个癌症代谢基因表达发生上调,3个癌症代谢基因表达发生下调.与CD133-细胞群相比,CD133+细胞群中糖酵解相关的基因有6个表达上调,colo205未分选细胞群的糖酵解相关的基因有7个表达轻微上调.葡糖转运蛋白1(glucose transporter 1,GLUT1)在CD133+细胞群中高表达,GLUT3在CD133-细胞群中高表达,GLUT4在未分选colo205细胞群高表达.参与三羧酸循环(tricarboxylic a c i d c y c l e)的6种癌症代谢基因中,谷氨酰胺酶1(glutaminase1,GLS1)在CD133-细胞群中高表达;异柠檬酸脱氢酶2(isocitrate dehydrogenase 2)和细胞色素氧化酶组装因子2(cytochromec oxidase assembly factors 2)在CD133-细胞群中低表达.共有20种癌症代谢调控基因在不同细胞群中发生一定程度表达变化.其中与colo205细胞群和CD133+细胞群相比,p53、RAS、细胞外调节蛋白激酶(extracellular regulated protein kinases)、肝脏激酶B1(liver kinase B1)、蛋白激酶B(protein kinase B)、TP53诱导的糖酵解及细胞凋亡调节蛋白(TP53-induced glycolysis and apoptosis regulator)在CD133-细胞群中均表达下调,而缺氧诱导因子1α(hypoxia inducible factor 1α)、IκB激酶(IκB kinase)和表皮生长因子受体(epidermal growth factor receptor)则表现上调.AMP激活的蛋白激酶(AMP-activated protein kinase)和生长因子受体酪氨酸激酶6(receptor tyrosine kinase)则在未分选细胞群中表达下调.结论:大肠癌colo205细胞株来源的CD133+、CD133-及colo205代谢基因及其调控基因表达存在着异质性,这可能对大肠癌的分子诊断、治疗靶点的选择和治疗手段的确定以及预后的判断提供参考.

AIM： To obtain differential gene expression information on cancer metabolic reprogramming from colo205 derived colorectal cancer cells.METHODS： Colo205 cell spheres were cultured in serum-free medium with cell factors, and CD133＋/CD133- cells were sorted using an indirect CD133 microbead kit. In vitro differentiation and nude mouse tumorigenicity assay were carried out to test whether CD133＋ cells have stem cell characteristics or not together with colo205 cells and CD133- cells. RNA-seq was employed for analysis of differential genes related to cancer metabolic reprogramming and metabolism regulatory genes, followed by verification by qRT-PCR.RESULTS： Compared with CD133- cells, 9 cancer metabolism related genes and 6 glycolysis related genes were up-regulated, and 3 cancer metabolism related genes downregulated in CD133＋ cells, while 12 cancer metabolism related genes and 6 glycolysis related genes were up-regulated, and 3 cancer metabolism related genes down-regulated in colo205 cells. For glucose transporters, glucose transporter 1（GLUT1） was up-regulated in CD133＋ cells, GLUT3 was up-regulated in CD133- cells, and GLUT4 was up-regulated in colo205 cells. Among the 6 cancer metabolism related genes involved in the tricarboxylic acid（TCA） cycle, glutaminase1（GLS1） was up-regulated and isocitrate dehydrogenase 2（IDH2） was down-regulated along with cytochromec oxidase assembly factors 2（SCO2） in CD133- cells. The mRNA expression levels of 20 cancer metabolism regulatory genes were changed, including p53, RAS, extracellular regulated protein kinases（ERK）, liver kinase B1（LKB1）, protein kinase B（AKT）, and TP53-induced glycolysis and apoptosis regulator（TIGAR） that were down-regulated, and hypoxia inducible factor 1α（HIF-1α）, IκB kinase（IKK） and epidermal growth factor receptor（EGFR） that were up-regulated in CD133-cells, and AMP-activated protein kinase（AMPK） and receptor tyrosine kinase（RTK6） that were down-regulated in colo205 cells.CONCLUSION： Heterogeneity of gene expression profile exists in colo205 cells and colo205 derived CD133- cells and CD133＋ cells, which may provide a reference for molecular diagnosis, therapeutic target selection, treatment evaluation and prognosis judgment in colorectal cancer.