基因芯片筛选人肺腺癌SPC-A-1干细胞样细胞特异性标志分子研究

Identification of specific markers in human lung adenocarcinoma cell line SPC-A1 stem-like cells using microarray

目的通过基因芯片技术筛选特异性的肺癌干细胞标志分子,为后续肺癌干细胞的筛选和功能研究奠定基础,为肺癌的发生机制和靶向治疗提供依据。方法将人肺腺癌SPC-A-1贴壁细胞(SPC-A-1monolayer cells,SPC-A-1-MC)作为对照组,将SPC-A-1-MC细胞培养于含20μg/L的重组人表皮生长因子(epidermal growth factor,EGF)和重组人碱性成纤维因子(basic fibroblast growth factor,b-FGF)及2%B27三种诱导因子的DMEM/F12培养基中诱导得到SPC-A-1干细胞样细胞(SPC-A-1stem-like cells,SPC-A-1-SC),作为实验组。收取两组细胞样本RNA,采用基因芯片技术筛选两组间差异表达基因,进一步对差异表达基因进行GO富集分析和KEGG通路富集分析。实时荧光定量PCR(quantitative real time polymerase chain reaction,qRT-PCR)验证部分关键差异表达基因在两组间的表达水平,采用两组独立样本t检验对两组间差异基因的表达结果进行统计分析。结果按照表达差异倍数|logFC|≥1.5且P<0.05的筛选条件,筛选出2094个差异表达基因,其中上调基因1069个,下调基因1025个,GO分析显示,差异表达基因主要参与组织发育、细胞对有机物的反应、细胞增殖凋亡调节、细胞外信号转导及对内源性刺激的反应过程,KEGG信号通路分析显示,主要涉及癌症相关通路、细胞色素P450代谢、类固醇激素合成的通路等。qRT-PCR验证部分差异表达基因,qRT-PCR验证结果与基因芯片结果一致。与SPC-A-1-MN组比较,SPC-A-1-SC组SPRY1上调(77.404±23.247)倍(t=-68.77,P<0.001),ANTXR2上调(17.454±1.163)倍(t=-34.22,P=0.001),SERPINE2上调(66.744±7.537)倍(t=-15.10,P<0.001),而KRT7下调(53.328±0.932)倍(t=69.70,P<0.001),DKK1下调(16.800±0.482)倍(t=199.37,P<0.001),两组间差异有统计学意义。结论通过基因芯片筛选肺腺癌干细胞样细胞与肺腺癌之间的差异表达基因,初步得出SPRY1、ANTXR2、SERPINE2、KRT7及DKK1有可能成为肺癌干细胞的特异性标记分子,为后续肺癌干细胞的筛选和功能研究奠定基础,同时可为肺癌的发生机制和靶向治疗提供实验依据。

OBJECTIVE This study screens specific lung cancer stem cell marker molecules by microarray technology,which lays the foundation for screening and functions of lung cancer stem cell,and provides a basis for the occurrence of lung cancer and targeted therapy.METHODS Human lung adenocarcinoma cell line SPC-A-1 attachment monolayer cells(SPC-A-1-MC)were selected as control group.SPC-A-1-MC cells were cultivated into DMEM/F12 medium containing three inducible factors as EGF,b-FGF and B27 to acquire SPC-A-1 stem-like cells(SPC-A-1-SC),and then were selected as experimental group.Acquire RNA was extracted from cell samples in two groups,and the differentially expressed genes between groups were identified by microarray,and differentially expressed genes were annotated by using Gene Ontology and KEGG signaling pathway enrichment.Moreover,the differentially expressed genes of SPRY1,ANTXR2,SERPINE2,KRT7 and DKK1 were further validated by quantitative real time polymerase chain reaction(qRT-PCR).Two independent sample t-tests were used to statistically analyze the expression results of differential genes between the two groups.RESULTS According to the screening conditions of expression difference factor|logFC|≥1.5 and P<0.05,2094 differentially expressed genes were obtained,including 1069 up-regulated genes and 1025 down-regulated genes.GO analysis showed that these differentially expressed genes were mainly correlated to tissue developement,cellular response to organic substance,regulation of cell proliferation and apoptosis,extracellular signal transduction,and response to endogenous stimuli.KEGG results shows that these genes were involved in pathways of cancer,metabolism of xenobiotics by cytochrome P450,and steroid hormone biosynthesis.Compared with SPC-A-1-MN group,SPRY1 was up-regulated(77.404±23.247)times,t=-68.77,P<0.001,ANTXR2 was up-regulated(17.454±1.163)times,t=-34.22,P=0.001 and SERPINE2 was up-regulated(66.744±7.537)times(t=-15.10,P<0.001)in SPC-A-1-SC group,but KRT7 was down-regulated(53.328±0.932)times(t=69.70,P<0.001),and DKK1 was down-regulated(16.800±0.482)times,t=199.37,P<0.001.CONCLUSION It is inferred that genes of SPRY1,ANTXR2,SERPINE2,KRT7 and DKK1 may become specific marker molecules for lung cancer stem cells.