Name: in vivo inhibition of microrna to decrease tumor growth in mice
Uploaded: 2019-08-23T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice at JoVE.com

我们感谢拉克尔·阿罗查·里斯科在老鼠的治疗和护理方面给予的帮助。我们感谢J.Blanco博士（加泰罗尼亚高级化学研究所-CSIC）和E.Mato博士（圣克里乌圣保医院研究所）巴塞罗那（西班牙）分别赠送CMV-萤火虫luc-IRES-EGFP和Cal62-Luc细胞。

动物实验是经欧洲委员会高级委员会道德委员会（CSIC，西班牙）批准，根据欧洲共同体法（86/609/EEC）和西班牙法律（R.D.1201/2005）进行的。
1. 细胞的侧角接种和肿瘤内性肿瘤治疗
<ol><li>细胞制备
	<ol>
<li>设计Cal62人类甲状腺癌细胞系（KRASG12R和p53A161D突变），以过度表达一种转基因结构，该结构构成表达GFP和荧光素酶（CMV-萤火虫Luc-IRES-eGFP）。通?...

<ol>
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本文介绍了一种研究miRNA体内功能的方法，以更好地了解其在肿瘤启动和进展中的作用，以及其作为甲状腺癌治疗靶点的潜力。这里描述的肿瘤异种移植模型基于细胞的使用，这些细胞可以通过其生物发光信号进行跟踪，从而允许在治疗的影响下测量体内的肿瘤生长。此外，我们描述了使用基于miRNA的甲状腺癌治疗，目前处于早期阶段。
我们首先使用皮下异种移植模型测试了m...

甲状腺癌是一种内分泌恶性肿瘤，发病率不断增加，虽然一般说来，它有很好的结果1。然而，一些患者发展为无法治疗的侵略性疾病，并且对分子基础知之甚少2。
miRNA 是 22 核苷酸长的非编码 RNA，用于调节许多组织中的基因表达，通常通过基对结合到目标信使RNA （mRNA） 的 3' 非转化区域 （3'UTR），触发 mRNA 退化或转化抑制3,4.越来越多的证据表明，微RNA表达的放松管制是癌症的标志，因为这些分子调节增殖信号、迁移、入侵和转移，并能抵抗凋亡5，6.近年来，许多研究已经确定miRNA是癌症诊断和预后的潜在生物标志物，以及治疗目标7，为癌症评价和治疗提供了新的维度。
miRNA在人类分子肿瘤学中已成为人类甲状腺肿瘤8、9、10、11、12的关键驱动因素。在miRNA向上调节， miR-146b是高度过度表达在皮皮脑甲状腺癌 （PTC） 肿瘤， 并证明显着显著增加细胞增殖， 并与侵略性和令人沮丧的预后6，12,13,14,15.此外，miR-146b调节与分化12有关的几个甲状腺基因，以及重要的肿瘤抑制基因，如PTEN16和DICER117。尽管miRNA疗法在癌症生物学中很重要，但仍处于早期阶段，很少有研究涉及甲状腺癌——最频繁的内分泌肿瘤18。在这里，我们描述了一种协议，使用两种不同的小鼠模型与人类衍生的肿瘤，其中，一个合成miRNA抑制剂（antagomiR）的分用，专门抑制细胞miRNA可以阻止肿瘤生长。我们首先使用一种常见的异种移植模型，而一种拮抗性肿瘤的局部肿瘤内管理降低了肿瘤的生长，测量为肿瘤生物发光16的减少。由于建立模仿人类肿瘤进展的强健小鼠模型对于开发独特的治疗方法至关重要，因此，原发性人类肿瘤的正射体植入是新药临床验证的更有价值的平台。皮下植入模型。因此，为了更好地评估拮抗性小鼠的治疗潜力，我们使用了在血流中系统传递的正交小鼠模型，获得了相同的结果。

<table>
	<tbody>
		<tr>
			<td>AntagomiR: mirVana miRNA inhibitor</td>
			<td>Thermo Fisher</td>
			<td>4464088</td>
			<td>In Vivo Ready</td>
		</tr>
		<tr>
			<td>Basement Membrane Matrix: Matrigel Basement Membrane Matrix High Concentration</td>
			<td>Corning</td>
			<td>#354248</td>
			<td></td>
		</tr>
		<tr>
			<td>DICER antibody</td>
			<td>Abcam</td>
			<td>ab14601</td>
			<td>IHQ: 1/100</td>
		</tr>
		<tr>
			<td>In vivo delivery reagent: Invivofectamine 3.0 Reagent</td>
			<td>Thermo Fisher</td>
			<td>IVF3005</td>
			<td></td>
		</tr>
		<tr>
			<td>In vivo imaging software: IVIS-Lumina II Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Negative control: mirVana miRNA Inhibitor, Negative Control #1</td>
			<td>Thermo Fisher</td>
			<td>4464077</td>
			<td>In Vivo Ready</td>
		</tr>
		<tr>
			<td>PCNA antibody</td>
			<td>Abcam</td>
			<td>ab92552</td>
			<td>WB: 1/2,000</td>
		</tr>
		<tr>
			<td>PTEN antibody</td>
			<td>Santa Cruz</td>
			<td>sc-7974</td>
			<td>WB: 1/1,000</td>
		</tr>
		<tr>
			<td>XenoLight D-Luciferin - K+ Salt Bioluminescent Substrate</td>
			<td>PerkinElmer</td>
			<td>122799</td>
			<td>Diluted in PBS</td>
		</tr>
	</tbody>
</table>

in vivo inhibition of microrna to decrease tumor growth in mice

微RNA（miRNA）是基因表达的重要调节器，通过它们破坏mRNA和抑制目标mRNA的翻译的能力。越来越多的研究已经确定miRNA是癌症诊断和治疗的潜在生物标志物，也是治疗目标，为癌症评估和治疗增加了一个额外的维度。在甲状腺癌的背景下，肿瘤的产生不仅来自重要基因的突变，还来自许多miRNA的过度表达。因此，miRNA在甲状腺基因表达控制中的作用正在演变为癌症的重要机制。本文提出一个方案，利用人类肿瘤异种移植和正交小鼠模型，研究miRNA抑制剂作为甲状腺癌治疗方式的效果。在工程稳定甲状腺肿瘤细胞表达GFP和荧光素酶后，细胞被注射到裸鼠中，形成肿瘤，随后可发生生物发光。体内抑制miRNA可以降低肿瘤生长，提高miRNA基因靶点。除了确定新的治疗靶点外，该方法还可用于评估确定 miRNA 在体内的重要性。

我们使用两个不同的小鼠模型来确定miRNA的中和是否能抑制肿瘤生长。因此，人类肿瘤甲状腺Cal62-luc细胞被分皮注射到裸鼠的侧翼，以生成异形文字模型。两周后，肿瘤成立，可以用卡钳测量。当时，小鼠在肿瘤内注射miR-146b抑制剂，或适当的对照，肿瘤体积再跟踪两周（图1A）。如图1B所示，在肿瘤内注射miR-146b抑制剂（抗146b）（n=8）的肿瘤生长对阴?...

Watch this Scientific Journal Video about In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice at JoVE.com

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

该协议将人类甲状腺肿瘤的异种移植和正交小鼠模型描述为测试基于微RNA的抑制剂治疗的平台。该方法是研究非编码RNA的功能及其作为新的治疗靶点的潜力的理想选择。

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

我们进行第一种治疗，基于甲状腺癌模型中的微RNA抑制剂。这些开发疗法为治疗这种疾病显示出希望。这种类型的正畸模型，使用系统的治疗传递途径，有助于验证新的微RNA药物。虽然我们使用正畸模型将人类甲状腺癌细胞植入小鼠甲状腺，但这些模型对于其他癌症类型可能是全身的。与朱莉娅·拉米雷斯-莫亚和阿德里安·阿库纳一起展示手术过程将是拉克尔·阿罗查·里埃斯科。她是我们研究所的技术员建立CAL-62人甲状腺癌细胞系不完全介质后，在4摄氏度的PBS的50微升中悬浮1倍10至第6细胞等同物，并混合与同等体积的地下室膜基质的细胞。将细胞装入1毫升注射器，配备27根量具半英寸针，皮下将100微升样品注射到6周大免疫缺陷阀C裸鼠的左侧侧翼。注射两周后，在1点5毫升管中加入160微升室温中室温的缓冲液或控制治疗缓冲液。并立即涡流溶液10秒，以确保混合物的复合。在50摄氏度下孵育治疗溶液30分钟，随后在微型离心机中短暂离心。然后，用新鲜的PBS和彻底的混合稀释沉淀处理复合物六倍。将整个200微升的治疗直接注射到肿瘤中。每周向每个实验动物注射50微升，每升40毫克D-路西芬基质溶液皮下2次。确保确认在异氟麻醉后对手趾捏缺乏反应。将动物放在体内生物发光成像系统的腔室中，根据标准协议使用体内成像软件对生物发光信号进行成像。然后分析肿瘤的生长。使用 T 检验比较两种治疗方法并确定显著性与生长差异。对于正畸甲状腺肿瘤细胞接种暂停在PBS的5微升中释放CAL-62细胞，并在皮下将100微升镇痛剂和100微升抗生素注射到7周大的瓣膜C裸鼠中。在确认对手趾捏没有反应后，将动物置于解剖显微镜下，用碘多莫酮对动物的脖子进行消毒。接下来，在皮肤上做一个大约2厘米的切口，并取代唾液的格兰斯暴露颈部。使用解剖钳和/或剪刀解剖带状肌肉以暴露气管和甲状腺，并使用 10 微升注射器，将 5 微升体积的肿瘤细胞注入位于十分软骨一侧的右甲状腺球。当所有细胞都已交付，重新定位唾液的牙，并使用丝绸编织，涂层，不可吸收的缝合线关闭切口。然后将碘多莫酮涂抹到伤口区域，将鼠标放在热毯上进行监测，直到完全恢复。在甲状腺内细胞注射两到三周后，如所证明的，准备治疗方案，通过麻醉甲状腺肿瘤携带动物静脉鼻窦的逆轨道注射，静脉注射。在这个具有代表性的实验中，肿瘤内注射无微RNA146B抑制剂的生长在负对方面受到显著抑制。与控制性肿瘤相比，在低水平、抗肿瘤治疗的肿瘤中，也观察到一些增殖标记的肿瘤内表达水平。此外，可以通过分析肿瘤提取的RNA或蛋白质来研究微RNA靶点的恢复。共同揭示个体微RNA的体内抑制是有效的，可用于甲状腺癌的治疗。如证明，对小鼠建立的甲状腺肿瘤进行组织学分析，可对肿瘤周围的上皮细胞进行染色，揭示肿瘤组织的甲状腺毛囊结构。值得注意的是，将微RNA146B抑制剂静脉注射到小鼠体内，与经过控制治疗的动物相比，肿瘤体积显著下降。此外，新描述的微RNA 146B靶在原发性肿瘤中的表达，在抗微RNA146B治疗后增加，进一步突出了抑制非原性微RNA表达的潜力，从而恢复其靶基因作为甲状腺癌的治疗。这项技术，以及随后的利平结果，为探索基于非固化RNA治疗甲状腺癌的新疗法提供了可能性。

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Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

将操纵的肿瘤相关中性粒细胞转移到肿瘤携带小鼠中,以研究其血管生成潜力在Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

研究癌症相关成纤维细胞在肿瘤生长中的作用的小鼠模型

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...