Name: discovery of driver genes in colorectal ht29-derived cancer stem-like tumorspheres
Uploaded: 2020-07-22T13:00:00
Description: Watch this Scientific Journal Video about Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres at JoVE.com

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。

1. 细胞培养和肿瘤球的形成
<ol><li>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）。</li>
	<li>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。</li>
	<li>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。</li>
	<li>使用血细胞计计算HT29细胞。</li>
...

<ol>
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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...

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。

<table>
	<tbody>
		<tr>
			<td>iRiS Digital Cell Imaging System</td>
			<td>Logos Biosystems, Inc</td>
			<td>I10999</td>
			<td>for observing the formation of tumorspheres</td>
		</tr>
		<tr>
			<td>Flow cytometry</td>
			<td>BD biosciences</td>
			<td>FACSCalibur</td>
			<td>for detecting the LGR5 and CD133 in the tumorspheres</td>
		</tr>
		<tr>
			<td>anti-LGR5-PE</td>
			<td>Biolegend</td>
			<td>373803</td>
			<td>LGR5 detection reagent</td>
		</tr>
		<tr>
			<td>anti-CD133-PE</td>
			<td>Biolegend</td>
			<td>372803</td>
			<td>CD133 detection reagent</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>GenScript</td>
			<td>Z00333</td>
			<td>for culture of tumorspheres</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>GenScript</td>
			<td>Z03116</td>
			<td>for culture of tumorspheres</td>
		</tr>
		<tr>
			<td>HGF</td>
			<td>GenScript</td>
			<td>Z03229</td>
			<td>for culture of tumorspheres</td>
		</tr>
		<tr>
			<td>IL6</td>
			<td>GenScript</td>
			<td>Z03034</td>
			<td>for culture of tumorspheres</td>
		</tr>
		<tr>
			<td>PureLink RNA extraction kit</td>
			<td>Invitrogen</td>
			<td>12183025</td>
			<td>isolate total RNA for RNAseq analysis</td>
		</tr>
		<tr>
			<td>RNAseq performance</td>
			<td>Biotools, Taiwan</td>
			<td></td>
			<td>RNAseq analysis is done commerially by Biotools, Ttaiwan</td>
		</tr>
		<tr>
			<td>NetworkAnalyst</td>
			<td>Institute of Parasitology, McGill University, Montreal, Quebec, Canada</td>
			<td></td>
			<td>http://www.networkanalyst.ca/</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>a statistical analysis software</td>
		</tr>
	</tbody>
</table>

discovery of driver genes in colorectal ht29-derived cancer stem-like tumorspheres

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。

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，...

Watch this Scientific Journal Video about Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres at JoVE.com

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

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。

大肠癌HT29衍生癌症干细胞类肿瘤球中的驱动基因发现

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

我们的协议利用RNA-Seq和生物信息学工具快速发现过度表达的驱动基因，这些基因有助于形成茎样结肠直肠肿瘤球。该协议用于筛选不同表达的基因和信号通路，以研究选择性结肠直肠癌中癌症干细胞的分子机制。在培养的第七天，使用带数字细胞成像系统的倒置显微镜来观察和测量肿瘤球的直径。当肿瘤球直径大于100微米时，用0.25%的胰腺素治疗培养。在37摄氏度下5分钟后，用生长介质体积的两倍中和尝试素，并使用血细胞计计算细胞。通过离心收集细胞。并在每100微升中浓度的4个细胞中，以5倍10的量重新暂停颗粒。在每个管中加入两个感兴趣的原抗体微升，在室温下孵育细胞30分钟，每分钟200转。在孵化结束时，用900微升PBS洗涤每个管，并分析细胞，通过流动细胞学表达感兴趣的蛋白质，根据标准协议。对于在胰蛋白酶采集后进行RNA分离，如刚刚证明的，每50微升PBS浓度将细胞重新以2倍10至第五细胞，用200微升的解液缓冲液将每个样品与每管两个微升的β·默卡托乙醇补充。在室温下短暂涡旋和五分钟孵育后，通过离心收集酸盐，并在每个管中加入200微升70%乙醇。将样品转移到快速柱中，通过离心去除溶剂。用400微升的洗涤液一和600微升的洗涤溶液洗涤样品，通过离心彻底去除任何非RNA，然后在同一离心机条件下再离心两分钟，以去除任何残留乙醇。然后将颗粒重新浸入每管50微升蒸馏水中，将样品离心1分钟，然后收集含有超自然物质的RNA。在RNA测序分析后，研究肿瘤球细胞与亲肿瘤细胞的差别基因表达。选择大于 1 到负 1 log2 折叠变化且读取计数大于 100 的基因在肿瘤圈和家长组中。并如前所述使用命令。获取火山图的数据，以确定差异基因表达。在网络分析员中，对于驱动基因选择，选择单个基因输入并复制并粘贴所选的过表达基因，将人类指定为生物体，官方基因符号指定为ID类型。单击"上传"并继续。要使用蛋白质-蛋白质相互作用来插入和分析数据，在遗传蛋白-蛋白质相互作用之后，使用S STRING相互作用组数据库，其置信度分数被切断900，以显示种子基因。交叉链接上传的基因。与更多单个基因相关的种子基因可以被选为驱动基因，这些基因可能参与维持肿瘤球的形成。在"布局"旋钮中的"背景图和强制图集"中选择"在映射概述中的白色"继续"。然后选择豹基对来分析上调节基因组。直径大于100微米的肿瘤球在培养后7天内形成。流量细胞测量分析显示，与亲亲HT29细胞相比，HT29衍生肿瘤球中的LGR5和CD133表达增加。RNA测序后，可以绘制出由热图确定的P值小于0.05的上、下调节值大于1对2倍的基因，以区分HT29衍生肿瘤球和亲亲HT29细胞之间的重要基因。在这个具有代表性的实验中，对超过1对2倍的上调节基因的分析产生了10个种子基因，将基因网络交叉连接。豹基对分析表明，两个基因与凋亡的负调控有关。此外，通过定量PCR验证了所选基因在HT29衍生肿瘤球中的表达。差异基因选择是程序中最重要的步骤。请记住，使用大于 1 的 log2 折叠更改作为标准，重新计票大于 100。使用此协议，从结肠直肠衍生肿瘤球中挑选的基因可以被击倒或过度表达，以发现它们在肿瘤球形成中的潜在作用。

Research

JoVE Journal

Cancer Research

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

肝大肠癌转移的高级动物模型：成像技术和转移性克隆性质

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

科研

癌症研究

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

遗传工程小鼠模型的散发性结肠直肠癌

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

寻找获得性抗靶治疗的驱动途径: 耐药亚克隆生成与基因击倒敏感性恢复

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

转移性结直肠癌患者循环微 rna 自定义面板的检测

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...