生物信息学分析肝癌干细胞中致瘤基因表达的差异

doi:10.3969/j.issn.2095-4344.2016.23.009

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
肝癌干细胞：具有CD90+，CD133+的肝癌细胞具有肿瘤干细胞的强增殖能力、克隆形成能力以及致瘤性特性，可以作为是肝癌肿瘤细胞的标志，而HepG2可以检测到两种细胞的表达。
基因芯片：又称 DNA 芯片、生物芯片。基因芯片技术系指将大量探针分子固定于支持物上后与标记的样品分子进行杂交，通过检测每个探针分子的杂交信号强度进而获取样品分子的数量和序列信息。通俗地说，就是通过微加工技术，将数以万计、乃至百万计的特定序列的DNA片段，有规律地排列固定于2 cm2的硅片、玻片等支持物上，构成的一个二维DNA探针阵列，与计算机的电子芯片十分相似，所以被称为基因芯片。

摘要
背景：研究认为，肝癌干细胞中致瘤基因表达与肝癌的发生、发展具有一定的关系，但目前尚缺乏对生物信息学和机制的研究。
目的：对于肝癌干细胞相关的致瘤性差异基因进行筛选，并对其进行生物信息学分析。
方法：将人肝癌细胞株HepG2作为肝癌干细胞的细胞工具制备总RNA并进行荧光标记，使用基因芯片进行杂交试验，将获得的mRNA表达谱进行筛选后获得差异性mRNAs，利用生物信息学进行GO注释和Pathway注释分析。
结果与结论：芯片杂交实验的检出率为73.21%，说明杂交试验是成功的；实验共有38 342个mRNA被发现，进一步分析后，筛选差异性表达基因1 236个(P < 0.05，fold change≥2)，其中上调和下调表达基因的数量分别为599和637个(P < 0.05)；表达差异基因的生物学功能主要涉及到了组蛋白的H4乙酰化，细胞的有丝分裂以及增殖、细胞相关蛋白质的合成以及分解、染色体的分离、细胞的分化以及凋亡、信号的转导、营养物质的运输、转录；Pathway注释主要涉及细胞因子介导的炎症信号通路、Wnt信号通路、Hedgehog通路以及Notch通路、TGF-Beta和Jak-STAT等。结果证实，肝癌干细胞中的差异表达基因与肝癌的发生有关，也可能参与信号通路的调控，这可为肝癌的治疗提供一种新靶点。

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
ORCID: 0000-0002-2029-4953(李淑娜)

关键词: 干细胞, 肿瘤干细胞, 肝癌, 致瘤基因, 生物信息学, 信号通路, 生物功能, 细胞培养, 差异基因, 基因芯片, 组织工程

Abstract:

BACKGROUND: Studies have suggested that oncogene expression in liver cancer stem cells has a certain relationship with the occurrence and development of liver cancer, but there is still a lack of research on bioinformatics and mechanisms.
OBJECTIVE: To identify differentially expressed genes in liver cancer stem cells and to analyze these genes by bioinformatics.
METHODS: The human hepatoma cell line HepG2 was the cell tool of liver cancer stem cells to prepare total RNA, and fluorescent labeling experiment was conducted. Using gene chips hybridization, mRNA expression profiles were obtained and were screened, and then differentially expressed mRNAs were obtained, GO and Pathway annotations were analyzed using bioinformatics.
RESULTS AND CONCLUSION: The detection rate was 73.21% for hybridization experiment, indicating the hybridization experiment is successful. In this study, a total of 38 342 mRNA were found, and after further analysis, 1 236 differentially expressed genes were screened (P < 0.05, fold change ≥ 2), including 599 up-regulated and 637 down-regulated genes, respectively (P < 0.05). Biological functions of differentially expressed genes were mainly involved in the histone H4 acetylation, cell mitosis and proliferation, synthesis and decomposition of cell-associated proteins, chromosome segregation, cell differentiation and apoptosis, signal transduction, as well as nutrient transportation and transcription. Pathway annotations were mainly related to cytokine-mediated inflammatory signaling pathway, Wnt signaling pathway, Hedgehog and Notch signaling pathways, TGF-Beta, Jak-STAT and so on. These results demonstrate that differentially expressed genes in liver cancer stem cells are related to the occurrence of liver cancer, and may be involved in the regulation of signaling pathways, which provides a new target for the treatment of liver cancer.

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: Stem cells, Liver Neoplasm, Tissue Engineering

中图分类号:

R394.2

李淑娜，宋娜娜. 生物信息学分析肝癌干细胞中致瘤基因表达的差异[J]. 中国组织工程研究, 2016, 20(23): 3407-3412.

Li Shu-na, Song Na-na . Bioinformatics analysis of differentially expressed oncogenes in liver cancer stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(23): 3407-3412.

图/表（结果） 1

2.1 总RNA的质量测定结果 经过紫外分光光度计检测发现，RAN的浓度约为0.45 mg/L，而A_{260 nm/280 nm}的比值为1.93-2.01；提取的总RNA经电泳检测结果发现， 18 S以及28 S电泳条带较亮，较为清晰，电泳带较为完整，未发现有明显的降解。

2.2 芯片杂交实验的检出率以及结果 进行3次杂交实验后，其平均杂交的检出率为73.21%，这一结果充分说明了本次的杂交试验是成功的，其芯片杂交图如图1所示：荧光信号分布较为均匀，信号点饱满，并且背景较低，进一步证明了杂交实验室成功的。图1是Cy3标记扫描图与Cy5标记扫描图叠加后得到的一个伪图，探针的颜色呈现黄色，表示大多数基因在两组间的表达是一致的。将原始数据导入到软件Feature Extraction software 10.7中进行分析，其中有38 342个mRNA被发现，进一步分析后，筛选差异性表达基因1 236个(P < 0.05，fold change ≥2)，其中上调和下调表达基因的数量分别为599和637个。这部分基因中有一部分与EMT差异表达有关。

2.3 生物信息学分析结果 对上述具有表达差异的基因863个已知基因进行Go注释分析结果发现，这些基因的生物学功能主要涉及到了组蛋白的H4乙酰化，细胞的有丝分裂以及增殖、细胞相关蛋白质的合成以及分解、染色体的分离、细胞的分化以及凋亡、信号的转导、营养物质的运输、转录，在肿瘤细胞分化以及自我更新过程，各种生物学过程均处于高度活跃状态。Pathway通路注释分析结果显示，基于KEGG数据库，信号通路表达差异基因主要涉及了与肝癌的途径和内吞作用相关信号通路，主要包括细胞因子介导的炎症信号通路、Wnt信号通路、Hedgehog通路以及Notch通路、TGF-Beta、Jak-STAT等。

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引言

材料方法

1.1 设计 差异蛋白基因生物信息学分析。

1.2 时间及地点 于2014年6月至2015年12月在中南大学湘雅医学院医学检验系实验室完成。

1.3 材料 实验使用的细胞株为人肝癌细胞株HepG2，购买于上海斯信生物科技有限公司。

1.4 实验方法

1.4.1 细胞培养 细胞株培养所使用的培养液为无血清培养液(含质量浓度20 μg/L表皮生长因子，20 μg/L碱性成纤维细胞生长因子，20 μg/L白血病抑制因子，体积分数2%B27，100 U/mL 青霉素，100 U/mL 链霉素的未加血清的DMEM/F12培养基)，用无血清培养液在超低黏附培养板中培养肝癌细胞，得到悬浮肝癌干细胞球。纯化后取成球细胞进行下一步实验。

1.4.2 RNA的抽取以及检测 使用普洛麦格(北京)生物技术有限公司的总RNA提取试剂盒并根据说明说中的标准流程提取细胞中的总RNA，抽取后使用美国Agilent公司生产的Agilent Bioanalyzer 2100生物芯片分析系统进行检验^[12]。检验合格后取4 μL总RNA样品，加无酶水至140 μL，稀释35倍后，使用美国Nanodrop公司的Nanodrop ND1000分光光度计测定样品在波长λ=260 nm 和λ=280 nm 的吸光度(A)值，计算RNA的总浓度以及纯度。用紫外线A_{260 nm/280 nm}表示RNA纯度，其比值在1.8-2.0则表示RNA纯度高。

1.4.3 基因芯片的杂交实验 参考以往方法^[13-14]，采用专门的表达谱芯片试剂盒对总RNA中的mRNA放大，并进行标记，然后使用RNeasymini kit将纯化cDNA纯化，具体的步骤应样严格参照试剂盒。于30 ℃条件下将cRNA样品片段化后，分别用用1.65 g Cy3、Cy5标记的cDNA与Agilent人全基因4*44K芯片，在65 ℃条件下，以10 r/min的速度滚动17 h进行杂交试验。

1.4.4 图像扫描及数据分析 杂交完成后用Agilent扫描仪扫描，并利用芯片结果报考，测定芯片检出率，确定杂交成功，利用Gene Spring Software 11.0对产异性基因进行分析。对筛选出的致瘤表达差异基因使用Gene Ontology数据库进行GO注释，采用基于超几何分布(hypergeometric distribution)的Fisher exact test进行计算每个GO的显著性水平(P值)，并对多重假设检验的结果进行校正并获得误判率(false discovery rate，FDR)，筛选出差异基因所体现的显著性GO。显著性筛选的标准：P < 0.01，上调和下调差异基因分别进行显著性功能分析，基于KEGG数据库进行Pathway注释。利用Fisher精确检验计算每个Pathway的显著性水平，并对多重假设检验的结果进行校正并获得FDR，筛选出差异基因所显著参与的pathway，显著性筛选的标准：P < 0.05。

1.5 主要观察指标 计算总RNA的纯度与浓度，通过Gene Ontology数据库进行GO注释分析；利用KEGG数据库进行信号转导通路分析。

讨论

目前，已经有研究表明，人类的肝癌组织或细胞系中存在着肝癌干细胞，其存在于肝癌的发生、发展、转移、术后复发以及治疗抗性等都有显著的相关性，因此，对肝癌干细胞的研究可能为肝癌的诊断以及靶向治疗提供新的方向，改善患者的预后^[15-17]。有研究很久以前就对肿瘤的发生与体内一群异质性细胞有密切的关系进行探讨，国外有研究者们成功的分离出了CD34⁺/CD38^-表型的人类肿瘤干细胞，首次证实了肿瘤干细胞在体内的存在^[18-19]。随着医学水平的不断进步，人们近几年来以及在多种癌症包括胰腺癌、前列腺癌以及胃癌等中发现了肿瘤细胞，进一步证明了研究者们关于肿瘤干细胞的学说^[20-23]。有研究发现，具有CD90⁺，CD133⁺的肝癌细胞具有肿瘤干细胞的强增殖能力、克隆形成能力以及致瘤性特性，可以作为是肝癌肿瘤细胞的标志，而HepG2可以检测到两种细胞的表达^[24-26]，因此，实验将HepG2细胞工具进行了干细胞的基因表达研究。

肝癌干细胞中的差异表达基因与肝癌的发生、发展以及预后有关系，也可能参与信号通路的调控^[18-22]。实验对HepG2中的总RNA进行了抽取与检测，结果发现总RNA的A_{260 nm/280 nm}的比值为1.93-2.01，电泳带较为完整，未发现有明显的降解，总RNA的纯度较高，质量完好，可以进行进一步的杂交实验。最近有相关研究报道，在肝癌产生之前，具有癌变可能的上皮细胞会在各种因素的影响下发生上皮-间质转化，从而使细胞内的基因发现中心的编排，获得了肿瘤侵袭能力以及干细胞性质，肝癌干细胞形成^[27-28]。实验的结果也发现，CD90⁺，CD133⁺高表达的细胞中，与上皮-间质转化相关的基因表达谱也明显的升高进一步证明了将CD90⁺，CD133⁺作为肝癌干细胞的标志是具有可靠性的。其次，实验的芯片杂交结果中发现了1 236个差异性表达基因，其中上调和下调表达基因的数量分别为599和637个，这些基因中有部分是与EMT相关的。文章对差异性表达基因进行了生物信息学分析，通过GO注释发现在肿瘤细胞分化以及自我更新过程中，各种生物学过程比如蛋白质的合成与分解、细胞的分化与凋亡，细胞有丝分裂以及增值等均处于高度活跃状态，这些过程可能参与了肝癌干细胞的发生于繁殖、转移^[29-30]。通过Pathway分析，与癌症途径相关的细胞因子介导的炎症信号通路、Wnt信号通路、Hedgehog通路以及Notch通路，TGF-Beta和Jak-STAT等均具有明显的基因差异性表达，实验认为这些通路均参与了肝癌干细胞的发生以及调控机制。通过生物信息学分析发现，影响肝癌干细胞产生的生物学行为以及信号通路是极为复杂的，但这些表达差异的基因具有一定的致瘤性，其可从整体上反映出肝癌干细胞的发生与分子调控机制^[31-58]。

综上所述，实验利用基因基因芯片技术筛选出的肝癌干细胞中的差异表达基因与肝癌的发生有关系，也可能参与信号通路的调控，这可以为肝癌的治疗提供一种新靶点。