Name: characterization of functionally associated mirnas in glioblastoma and their engineering into artificial clusters for gene therapy
Uploaded: 2019-10-04T14:30:00
Description: Watch this Scientific Journal Video about Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy at JoVE.com

作者希望感谢哈维库欣神经肿瘤学实验室的成员的支持和建设性的批评。这项工作得到了NINDS授予K12NS80223和K08NS101091给P.P.P.的支持。

1. 胶质母细胞瘤中功能相关miRNA的特征
<ol><li>胶质细胞瘤与大脑广泛差分miRNA表达的分析
	<ol>
<li>首先，确定肿瘤中最显著解除调节的miRNA。这可以通过至少三种不同的方法实现：<ol>
<li>挖掘癌症基因组图谱，发现https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga，测序数据11。</li>
			<li>从新鲜操作样本12执行微阵列分析。</li>
			<li>使?...

<ol>
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该协议基于这样一种概念，即miRNA不是孤立地运作，而是通过小组工作在生物学上相关，这些组是由特定的细胞上下文26转录确定的。为了从翻译的角度证明这种方法的合理性，引入了一种后续协议，允许在细胞/组织中重新使用这种多miRNA模式。这是可以利用miRNA相对简单的生物发生，通过微处理器识别特征miRNA发夹是必要的，并足以正确miRNA处理27。同时，这一?...

miRNA为开发一种广泛的基因治疗方法提供了无可比拟的机会，治疗许多疾病1、2、3，包括癌症4，5。这是基于这些生物分子的几个独特特征，包括其小尺寸6，简单的生物成因7，和自然倾向在关联8中发挥作用。许多疾病的特点是特定的miRNA表达模式，这往往收敛于复杂生物功能的调节9。此方法的目的是首先定义一个策略，以确定与特定细胞功能协同相关的 miRNA 组。因此，它为在下游研究和应用中重新建立这种miRNA组合提供了一个策略。
该方法允许同时对多个miRNA进行功能分析，同时针对大量mRNA，从而概括疾病的复杂景观。这种方法最近被用来定义一组三miRNA，其中1）同时在脑癌中降低调节，2）在神经分化期间表现出强烈的共表达模式，以及对辐射或脱氧核糖核酸烷基化剂。下面描述的聚类方法对三个miRNA模块的这一模块的重新表达，对癌细胞的生物学产生了深刻的干扰，并可以很容易地用作临床前研究的基因治疗策略。该协议可能特别感兴趣的那些参与miRNA研究及其翻译应用。

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	<tbody>
		<tr>
			<td>0.4% low melting temperature agarose&#160;</td>
			<td>IBI Scientific</td>
			<td>IB70058</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;M sterile filter unit</td>
			<td>Merck Millipore</td>
			<td>SLH033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5-mL Microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well plates&#160;</td>
			<td>Greiner Bio-One</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Athymic mice (FoxN1 nu/nu)</td>
			<td>Envigo</td>
			<td>069(nu)/070(nu/+)</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>Greiner Bio-One</td>
			<td>660175</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper, 16cm</td>
			<td>Sarstedt</td>
			<td>83.1832</td>
			<td></td>
		</tr>
		<tr>
			<td>Cesium 137 irradiator&#160;</td>
			<td>JL Sheperd and Associates</td>
			<td>Core Facility (Harvard Medical School)</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>439142-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline&#160;</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-293</td>
			<td>American Type Culture Collecti</td>
			<td>ATCC CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Sigma-Aldrich</td>
			<td>1051750500</td>
			<td></td>
		</tr>
		<tr>
			<td>Human primary glioma stem-like cells (GBM62)</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. E. A. Chiocca (Brigham and Women&#8217;s Hospital, Boston, MA)</td>
		</tr>
		<tr>
			<td>Human primary glioma stem-like cells (MGG4)</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. Hiroaki Wakimoto (Massachusetts General Hospital, Boston, MA)</td>
		</tr>
		<tr>
			<td>Lentiviral vector pCDH-CMV-MCS-EF1-copGFP</td>
			<td>System Biosciences</td>
			<td>CD511B-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge refrigerated</td>
			<td>Eppendorf</td>
			<td>model no. 5424 R, cat. no.5404000138</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4112APG</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes&#160;</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>3117-0380PK</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>2000c</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Progenitor cells (NPC)</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. Jakub Godlewski (Brigham and Women&#8217;s Hospital, Boston, MA)</td>
		</tr>
		<tr>
			<td>Neurobasal Medium&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon eclipse Ti motorized fluorescent microscope system</td>
			<td>Nikon, Japan</td>
			<td>14314</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985088</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>CLS6571-960EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-Dishes 94/16&#160;</td>
			<td>Greiner Bio-One</td>
			<td>632180</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine&#160;</td>
			<td>Sigma- Aldrich</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF&#160;</td>
			<td>PeproTech&#160;</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic&#160;</td>
			<td>PeproTech&#160;</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Gibco</td>
			<td>12587-010&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Miniprep Kit</td>
			<td>Direct-zol</td>
			<td>R2050</td>
			<td></td>
		</tr>
		<tr>
			<td>S1000 Thermal Cycler&#160;</td>
			<td>Bio-Rad</td>
			<td>1852196</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Image-Guided Micro Irradiator&#160;</td>
			<td>Xstrahal Life Sciences, UK</td>
			<td>Core facility (Dana-Farber Cancer Institute, Boston, MA)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall WX+ Ultracentrifuge&#160;</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>75000100</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOne Real-Time PCR System</td>
			<td>Applied Biosystems&#160;</td>
			<td>4376357</td>
			<td></td>
		</tr>
		<tr>
			<td>SterilGARD biosafety cabinet&#160;</td>
			<td>The Baker Company</td>
			<td>SG403A-HE</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>T98-G</td>
			<td>American Type Culture Collecti</td>
			<td>ATCC CRL-1690</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4366596</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4324018</td>
			<td></td>
		</tr>
		<tr>
			<td>Temozolomide</td>
			<td>Tocris Bioscience</td>
			<td>2706</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek optimum cutting temperature&#160;</td>
			<td>Fisher Scientific</td>
			<td>NC9636948</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td>Lysis reagent</td>
		</tr>
		<tr>
			<td>U251-MG</td>
			<td>American Type Culture Collecti</td>
			<td>ATCC HTB-17</td>
			<td></td>
		</tr>
		<tr>
			<td>U87-MG&#160;</td>
			<td>American Type Culture Collecti</td>
			<td>ATCC HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td>ViraPower Lentivector Expression system&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>K4970-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, HPLC grade</td>
			<td>Fisher</td>
			<td>W54</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of functionally associated mirnas in glioblastoma and their engineering into artificial clusters for gene therapy

微RNA（miRNA）在健康和疾病中的生物学相关性明显依赖于许多同时解除调节的miRNA的特定组合，而不是单个miRNA的作用。这些特定的miRNA模块的表征是最大化其在治疗中的应用的一个基本步骤。这非常相关，因为它们的组合属性实际上可以加以利用。此处描述的方法定义与胶质细胞瘤中致癌染色质抑制器控制相关的特定 miRNA 签名。该方法首先定义了一般组miRNA，与正常组织相比，在肿瘤中解除调节。分析通过不同的培养条件得到进一步改进，强调在特定细胞状态下同时共同表达的miRNA子组。最后，满足这些滤光片的miRNA被组合成一个人工多cistronic转基因，该基因基于自然存在的miRNA簇基因的脚手架，然后用于将这些miRNA模块过度表达到接收细胞中。

这种方法允许对三个miRNA模块进行表征，这些模块在脑肿瘤中一直向下调节，在神经元分化期间具体表达（图1），并参与后肿瘤生存反应治疗 （图 2）。这是通过调节复杂的致病染色质抑制途径来实现的。这种共表达模式表明这三个miRNA之间有很强的协同活性（图3）。因此，利用miRNA的体积小和简单的生?...

Watch this Scientific Journal Video about Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy at JoVE.com

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

此处描述的是一种将生物协同miRNA模块及其组装成短转基因的模块的特征，允许基因治疗应用同时过度表达。

胶质母细胞瘤中功能相关miRNA的特性及其工程在基因治疗的人工簇中

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

该协议允许在真核细胞中同时调制许多微RNA，并且基于一种非常简单和灵活的方法，可最大限度地减少分子克隆步骤。这项技术对研究体外微RNA功能相互作用有影响，但更重要的是，它为在治疗中更有效地使用微RNA提供了一个平台。我们主要在脑癌的背景下开发和使用它，但它适用于任何涉及基因表达的多重改变的疾病。我们已经在其他模型中测试了我们的人工转基因，如乳腺癌和甲状腺癌线，显示这种技术的可重复性。生物信息学和微RNA生物学的基本知识有助于此协议，但不是不可或缺的。生物信息学方法对于定义相关的微RNA组合和TRK-11非常重要，而熟悉微RNA处理对于了解如何将这些不同的微RNA组装成人工DNA簇非常重要，这些DNA在稳定到特定细胞后可以成功处理。要涂上盘子，首先以每毫升100微克的浓度将聚D-lysine溶解在水中。每 25 平方厘米表面以 1 毫升溶液将溶液倒入盘中。旋转的菜，以确保覆盖整个菜。六小时后吸气聚D-晶酸溶液，并用PBS冲洗两次。板人神经干细胞在5毫升的介质中，无论是干细胞培养基、星细胞分化培养基还是神经元分化培养基，都相当于10万个细胞。细胞电镀后，将培养皿放在37摄氏度和5%的二氧化碳培养箱中一周，然后根据手稿进行微RNA表达分析。要培养GBM-34胶质母细胞瘤干细胞样细胞，从1倍十到第五细胞和5毫升神经基础培养基开始，在25平方厘米的低附着烧瓶中。将烧瓶放入摄氏37度和5%的二氧化碳孵化器中。电镀后48小时，每7天加入双倍的DNA氧化硅剂特莫佐洛米德浓度。首先添加五微摩尔TMZ，在五毫升介质中补充五天。五天后，取出介质，代之以没有特莫佐洛米德的新鲜介质，使存活的细胞恢复。48小时后，加入10微摩尔特莫佐洛米德，再孵育5天。重复双倍的特莫佐洛米德治疗，直到达到至少100微摩尔的浓度，细胞对药物产生抗药性。诱导电阻后，使用1毫升的Lysis试剂说谎细胞。提取总RNA，根据手稿分析特定微RNA的表达。要获得先前定义的每个微RNA的预测靶位，请使用微RNA靶向预测工具。从 TargetScan 的头版，从预先填充的下拉菜单中选择感兴趣的微 RNA。单击提交按钮。使用下载表链接将生成的目标列表下载为电子表格。为了减少误报预测的机会，请仅将目标包括在保存的站点列和下游分析中。其他微RNA预测程序获得进一步的串联性，并且只包括所有算法都通用的目标。然后，打开托普金套房 20。为了评估每个微RNA常见的通路的丰富性，粘贴在训练基因集窗口中获得的目标列表。单击 HGNC 符号作为输入类型。单击提交并开始。该程序提供了一个输出表，显示输入的基因列表最重要的 GO 类别。要最终确定每个微RNA对公共通路或细胞过程的调节的贡献，请打开生物信息学和进化基因组学网站提供的文图功能。根据特定细胞过程所涉及的基因的完整列表检查每个获得的目标组。如果每个感兴趣的微型RNA的目标信使RNA都在各自的窗口中，则上传复制粘贴列表。使用唯一标识符命名每个列表。单击"提交"。该程序提供一个文图的可视化输出，包含每个扇区的基因数，以及每个子集和交点组合的信使RNA的完整列表。要将选定的微RNA分组到功能性转基因中，获得基于miR-17-92聚类位点的转基因基架。从组合基因组浏览器获取核苷酸序列。选择大约 800 个碱基对核心序列，包括该位点的所有六个编码的微RNA发夹和核心序列的上游和下游至少 200 个核苷酸侧翼序列。将序列粘贴到任何单词编辑程序中。通过以 miR 基检索六个本机毛针的顺序，定义每个毛针的顺序。在以前标识的核心序列范围内标记每个序列。每个发夹之间的任何序列表示分位器序列。接下来，从miR基获取在转基因中过度表达的微RNA的发夹序列。仔细注意微处理器裂解位点中的特定核苷酸。从核心序列中删除本机发夹序列，但每个发夹的五端和末端的三到五个核苷酸除外，这些核苷酸将作为新发夹的接受器。粘贴所需微 RNA 的发夹序列，以替换以前删除的发夹。在转基因的两个侧翼区域添加所需的限制序列，以方便子克隆进入选择的传递载体。验证所选限制序列本身中不存在。为了验证转基因的二维结构，将完整的转基因序列复制到RNA结构预测软件程序RNA Webfold中。选择标准程序设置，然后单击"继续"。分析图形输出，特别是对于存在至少 11 个核苷酸与微处理器裂解位点有双绞合茎结构存在明确定义的发夹。此外，查找发夹序列中缺少分支点。此时，转基因已准备好通过基因合成产生，并克隆到所需的传递载体中。这种方法允许对三个微型RNA的模块进行表征，这些RNA在脑肿瘤中一直受到向下调节，在神经元分化期间特别共同表达。在基因毒性应激期间，微RNA模块的共表达模式得到确认，并暗示了这三个微RNA之间的强协同活性。设计的转基因可以同时重述三个微RNA在体外和体内细胞中的表达，对肿瘤生物学有重大干扰，并有望实现转化适用性。此外，转基因集群在乳腺癌模型中也发挥作用。该协议的关键要求是保持转基因中处理器裂解的原始大小。并确保保持嵌合微RNA发夹的茎环结构。通过这种方法，我们可以将多个生物活性微RNA基因浓缩成非常短的DNA序列，这些DNA序列可以真正地安装在任何基因治疗的传递载体中。此方法是研究不同微RNA之间的功能相互作用和干扰复杂的多因素细胞通路，否则将需要多种药物组合的理想方法。

Research

JoVE Journal

Genetics

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

科研

遗传学

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Production and Purification of Baculovirus for Gene Therapy Application

杆状病毒基因治疗应用的生产与纯化

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

非人类灵长类动物造血干细胞和祖细胞的制备及基因修饰

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...