大肠癌HT29衍生癌症干细胞类肿瘤球中的驱动基因发现
1Radiation Biology Research Center, Institute for Radiological Research,Chang Gung University, Chang Gung Memorial Hospital at Linkou, 2Department of Medical Imaging and Radiological Sciences,Chang Gung University, 3Department of Radiation Oncology,Chang Gung Memorial Hospital, 4Department of Laboratory Medicine,Mackay Memorial Hospital
Summary
这里介绍的是一个协议，用于发现过度表达的驱动基因，维持从结肠直肠HT29细胞中提取的癌症干细胞样细胞。RNAseq 与可用的生物信息学执行调查和筛选基因表达网络，以阐明参与目标肿瘤细胞生存的潜在机制。
Abstract
癌症干细胞对临床治疗起着至关重要的作用，导致肿瘤复发。有许多肿瘤基因与肿瘤的发生和癌症干细胞特性的启动有关。由于结直肠癌衍生肿瘤球形成中的基因表达尚不清楚，因此需要时间来发现一次对一个基因进行工作的机制。这项研究演示了一种快速发现参与体外结直肠癌干细胞存活的驱动基因的方法。在本研究中选择并使用了用于表达LGR5的结肠直肠HT29癌细胞，这些癌细胞在培养为球类时伴随增加CD133茎度标记。所提出的协议用于使用可用的生物信息学执行RNAseq，以快速发现在结直肠HT29衍生的茎类肿瘤球的形成中过度表达的驱动基因。该方法可以快速筛选和发现其他疾病模型中的潜在驱动基因。
Introduction
结肠直肠癌（CRC）是导致全球高患病率和高死亡率的主要原因,。由于基因突变和扩增，癌细胞生长没有增殖控制，这有助于细胞生存3，抗凋亡4，癌症干细胞5,5，6，7。,7在肿瘤组织内，肿瘤异质性使肿瘤细胞在治疗期间能够适应和存活。癌症干细胞（CSCs）自我更新率和多能性高于差异性癌症类型，主要负责肿瘤复发9、10,10和转移性CRC11。CSC具有更多的耐药性12，13，14和抗凋亡的12,13,14特性15，16，16从而生存肿瘤化疗。
在这里，为了研究所选CRC干细胞中干细胞的潜在机理，RNAseq对肿瘤球体中不同表达的基因进行了筛选。癌细胞在低粘附条件下生长，受培养基（包括EGF、bFGF、HGF和IL6）的生长因子刺激时，可以形成球状体（也称为肿瘤球）。因此，我们选择了CRC HT29肿瘤细胞，在用氧化铂和伊里诺特康17治疗时，抗化疗的磷酸化STAT3增加。此外，HT29 在所述培养条件下培养时表示较高的干性标记。HT29衍生的CSC模型表示高量的白化素富含重复性G蛋白耦合受体5（LGR5）18，CRC干细胞18的特定标记19，20。20此外，CD133，被认为是癌症干细胞的一般生物标志物，在HT29细胞系21中也高度表达。该协议的目的是发现基于生物信息学数据集的既定癌症干细胞类肿瘤球中的驱动基因组，而不是研究个体肿瘤基因22。它通过RNAseq分析研究潜在的分子机制，然后进行现有的生物信息学分析。
下一代测序是一种高通量、易用、可靠的DNA测序方法，基于计算帮助，用于全面筛选驱动基因，指导肿瘤治疗23。该技术还用于检测基因表达从逆转录的分离的RNA样本24。然而，当使用RNAseq进行筛选时，以治疗为目标最重要的基因在实验样本和控制样本之间可能没有最高的表达差异。因此，一些生物信息学被开发用于根据当前数据集（如KEGG25、GO26、2726,或黑豹28）对基因进行分类和识别，包括独创性通路分析（IPA）29和网络分析2930。该协议显示 RNAseq 和 NetworkAnalyst 的集成，以快速发现与亲 HT29 细胞相比，所选 HT29 衍生球体中的一组基因。建议将这种方法应用到其他疾病模型，以发现重要基因的差异。
与个体基因表达研究相比，高通量技术为肿瘤精密医学寻找潜在驱动基因提供了优势。借助 KEGG、GO 或 PANTHER 等有用数据集，可以根据疾病模型、信号通路或特定功能识别特定基因，从而快速关注特定的重要基因，从而节省时间和研究成本。类似的应用在以前的研究14，18，31,18,使用。特别是，肿瘤是更复杂的，因为不同类型的肿瘤表达区分基因和生存和增殖的途径。因此，本协议可以拾取不同情况下区分不同肿瘤类型的基因。通过了解特定基因表达的机制，有可能找到有效的癌症策略。
Protocol
1. 细胞培养和肿瘤球的形成 培养HT29细胞在10厘米的培养皿含有Dulbecco的改良鹰介质（DMEM）与10%的胎儿牛血清（FBS）和1%青霉素链霉素抗生素（P/S）。 在37°C的培养箱中生长细胞，在无菌条件下湿度为5%CO2和95%，直到达到80%的汇合。 在37°C下用1 mL 0.25% trypsin对HT29细胞进行试穿，5分钟，从而通过添加2 mL的DMEM和1%的P/S来中和尝试。 使用血细胞计计算HT29细胞。 …
Representative Results
为了建立研究癌症干细胞机制的模型，将结肠直肠HT29细胞用于在含有B27、EGF、bFGF、HGF和IL6的低附板中体外培养癌症干细胞类肿瘤球。肿瘤球直径为100μm，在7天内形成（图1A）。肿瘤球被胰腺素化为单细胞，并使用流式细胞学检测LGR5和CD133表达进行分析。LGR5在HT29驱动肿瘤球中从1.1%增加到11.4%，并且使用流细胞学检测细胞（图1B）。与亲亲HT29细胞相比，…
Discussion
在这项研究中，培养的癌症干细胞样肿瘤球被用作用可用的生物信息学分析RNAseq数据的模型。对于疾病模型，使用了HT29衍生的肿瘤球。由于肿瘤球对肿瘤疗法具有耐药性，因此利用建立的模型通过研究基因表达的差异来研究抗药性的详细机制。此外，使用RNAseq与可用的生物信息学的基因组技术提供了快速了解的研究模型，因此可能涉及的基因可以更有信心地验证。此外，可以识别肿瘤球形成中涉…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢张群纪念医院放射研究所放射生物学核心实验室的技术支持。这项研究得到了长贡纪念医院（CMRPD1J0321）、成新综合医院（CHGH 106-06）和麦凯纪念医院（MMH-CT-10605和MMH-106-61）的资助。供资机构在设计和数据收集、分析和解释数据或撰写手稿方面没有任何影响。
Materials
| iRiS Digital Cell Imaging System | Logos Biosystems, Inc | I10999 | for observing the formation of tumorspheres |
| Flow cytometry | BD biosciences | FACSCalibur | for detecting the LGR5 and CD133 in the tumorspheres |
| anti-LGR5-PE | Biolegend | 373803 | LGR5 detection reagent |
| anti-CD133-PE | Biolegend | 372803 | CD133 detection reagent |
| EGF | GenScript | Z00333 | for culture of tumorspheres |
| bFGF | GenScript | Z03116 | for culture of tumorspheres |
| HGF | GenScript | Z03229 | for culture of tumorspheres |
| IL6 | GenScript | Z03034 | for culture of tumorspheres |
| PureLink RNA extraction kit | Invitrogen | 12183025 | isolate total RNA for RNAseq analysis |
| RNAseq performance | Biotools, Taiwan | RNAseq analysis is done commerially by Biotools, Ttaiwan | |
| NetworkAnalyst | Institute of Parasitology, McGill University, Montreal, Quebec, Canada | http://www.networkanalyst.ca/ | |
| Prism | GraphPad Software | a statistical analysis software |
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Cite This Article
Cheng, C., Hsu, P., Sie, Z., Chen, F. Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres. J. Vis. Exp. (161), e61077, doi:10.3791/61077 (2020).