Genome-wide transcriptional profiling of NB4 leukemic cells affected by umbilical cord-derived mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.25.003

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells (MSCs) are an important component of the in vivo microenvironment and act on multiple biological behaviors of tumor cells. The potential clinical value of MSCs has become an issue of concern in recent years.
OBJECTIVE: To investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with umbilical cord-derived MSCs (UC-MSCs) using cDNA microarray.
METHODS: In vitro co-culture system was constructed, and then cellular proliferation, apoptosis and differentiation status of NB4 cells treated with UC-MSCs were evaluated. Two cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without UC-MSCs. The probes were labeled with fluorescence dyes individually, hybridized with cDNA microarray, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in two gene expression profiles.
RESULTS AND CONCLUSION: UC-MSCs promoted the proliferation and differentiation, while reduced the apoptosis of NB4 cells. The analysis of gene expression profiles indicated that after co-culture with UC-MSCs, 530 genes were up-regulated and 53 genes were down-regulated. Accordingly, specific gene function and pathway signaling related were also regulated to some extent. Overall, UC-MSCs influence can major biological behaviors of NB4 cells by changing expression of a large amount of genes, gene-related function and multiple intracellular signaling pathways.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Mesenchymal Stem Cells, Leukemia, Promyelocytic, Acute, Microchip Analytical Procedures, Tissue Engineering

CLC Number:

R394.2

Fan Hui-fang, Chen Fang, Ma Feng-xia, Chi Ying, Lu Shi-hong, Han Zhong-chao. Genome-wide transcriptional profiling of NB4 leukemic cells affected by umbilical cord-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3949-3955.

Figures/Tables 3

2.1 UC-MSCs影响NB4白血病细胞的生物学行为共培养72 h后检测细胞增殖，UC-MSCs促进白血病细胞的生长(图1A)；共培养48 h后检测细胞凋亡，UC-MSCs抑制5 μmol/L阿糖胞苷诱导的NB4细胞的凋亡(图1B)；共培养72 h在显微镜下观察瑞士吉姆萨染色结果，并且于24，48，72 h分别检测细胞表面粒系分化抗原CD11b表达，NB4细胞出现了向粒细胞分化的特征(图1C和1D)。 2.2 通过随机方差模型(RVM)筛选差异基因与NB4单独培养对照组相比，NB4+UC-MSCs共培养组的NB4细胞共有583个基因出现了明显的差异表达(P < 0.05，FDR < 0.05)，其中530个基因上调，53个基因下调(图2A)。一些通常被认为与细胞增殖、凋亡、分化和免疫反应相关的基因如BCAT1，MCL-1，ITGAM，ITGAX，CEBPB，ICAM1，CD58，TLR4，TLR6，CCL2，IL-8等基因的表达分别增加了1.66倍，1.44倍，1.73倍，1.95倍，2.45倍，2.35倍，1.39倍，1.97倍，1.92倍，18.25倍和2.53倍(图2B)。 2.3 差异表达基因的GO分析通过GO数据库对差异表达基因进行了GO分析，并通过双侧Fisher’s精确检验和χ2 检验评估数据的意义[13，18]。P < 0.01被认为差异有显著性意义。-LgP代表了调节显著程度。UC-MSCs作用下，上调最明显的5个GO分别为白细胞迁移(leukocyte migration)，凝血功能(blood coagulation)，炎症反应(inflammatory response)，细胞信号转导(signal transduction)和离子转运(ion transport)，见图3A。另外，整合素介导的信号通路(integrin-mediated signaling pathway)，中性粒细胞趋化(neutrophil chemotaxis)，细胞形态调节(regulation of cell shape)，固有免疫应答(innate immune response)，抗凋亡(anti-apoptosis)，整合素介导的细胞黏附(cell adhesion mediated by integrin)，免疫反应中中性粒细胞的激活(neutrophil activation involved in immune response)，细胞增殖(cell proliferation)，呼吸爆发(respiratory burst)和细胞成熟(cell maturation)相关的GO亦有明显上调。UC-MSCs作用下，下调的GO很少，下调最明显的5个GO分别是呼吸电子传递链(respiratory electron transport chain)，细胞对锌离子的反应(cellular response to zinc ion)，生长负调控(negative regulation of growth)，视泡的形态发生(optic vesicle morphogenesis)和视杯的结构组织(optic cup structural organization)，见图3B。 2.4 GO-Map分析根据GO map能直接并系统地发现基因功能之间的网络关系。如图4所示，基因功能调节主要表现在4个方面：①免疫反应(immune response)：免疫反应的调节(regulation of immune response)，炎症反应(inflammatory response)和吞噬作用(phagocytosis)；②细胞生理(Cellular process)：细胞成熟(cell maturation)、迁移(cell migration)和黏附(cell adhesion)；③对刺激的反应(response to stimuli)：对低氧的反应(response to hypoxia)，内质网应激(response to endoplasmic reticulum stress)和细胞对脂多糖的反应(cellular response to lipopolysaccharide)；④细胞信号(cell signaling)：信号转导(signal transduction)和蛋白磷酸化(protein phosphorylation)。 2.5 Pathway分析根据KEGG数据库，分析了差异表达基因相关的信号通路，如图5所示，UC-MSCs作用下最有意义的通路是白细胞跨内皮迁移(leukocyte transendothelial migration)，肿瘤转录失调(transcriptional misregulation in cancers)，细胞黏附分子(CAMs)，Fcγ受体介导的吞噬作用(Fc gamma-R mediated phagocytosis)和肌动蛋白骨架的调节(regulation of actin skeleton)。这些通路与中性粒细胞的激活关系密切，代表了中性粒细胞功能的成熟和形态的变化。 2.6 Path-Net分析根据KEGG数据库进行了Path-Net分析。如图6所示，通路相互作用主要集中在斑状黏着(focal adhesion)，肿瘤信号通路(pathways in cancer)和细胞因子-细胞因子受体相互作用(cytokine-cytokine receptor interaction)。另外还有氨基酸代谢网络(amino acid metabolism network)和肌动蛋白细胞骨架调节(regulation of actin cytoskeleton)网络。用“度(degree)”去评估通路的相互作用。表1列出了有意义的通路。斑状黏着(focal adhesion)(degree=6)和肿瘤信号通路(pathways in cancer)(degree=6)的degree最高，这预示着这两个信号通路在UC-MSCs对NB4细胞的影响上发挥了重要作用。 2.7 RT-PCR验证差异基因表达选择数个功能密切相关的基因通过RT-PCR方法验证芯片分析的可信性。IL-8，TLR6，CCL2，ITGAM，ITGAX，CEBPB，CD54，MCL-1 和 BCAT1与主要的细胞生物学表现相关，对这几个基因进行了RT-PCR分析。基因芯片中上述基因的mRNA表达分别增加了2.53 倍，1.92倍，18.25倍，1.73倍，1.95倍，2.45倍，2.35倍，1.44倍和1.66 倍。RT-PCR数据与芯片结果一致，证实了与单独培养NB4细胞相比，这些基因在与UC-MSCs共培养组的NB4细胞中确实存在明显的上调(图7)。"

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