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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 胶质母细胞瘤中功能相关miRNA的特征 胶质细胞瘤与大脑广泛差分miRNA表达的分析 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： 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。 从新鲜操作样本12执行微阵列分析。 使?…
Representative Results
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?…
Discussion
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?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.4% low melting temperature agarose | IBI Scientific | IB70058 | |
| 0.45 µM sterile filter unit | Merck Millipore | SLH033RS | |
| 1.5-mL Microcentrifuge tube | Eppendorf | 22431081 | |
| 6-Well plates | Greiner Bio-One | 657160 | |
| Athymic mice (FoxN1 nu/nu) | Envigo | 069(nu)/070(nu/+) | |
| B-27 Supplement | Thermo Fisher Scientific | 12587010 | |
| Cell culture flask | Greiner Bio-One | 660175 | |
| Cell Scraper, 16cm | Sarstedt | 83.1832 | |
| Cesium 137 irradiator | JL Sheperd and Associates | Core Facility (Harvard Medical School) | |
| Chloroform | Sigma-Aldrich | 439142-4L | |
| DMEM, high glucose, pyruvate | Thermo Fisher Scientific | 11995040 | |
| Dulbecco’s phosphate-buffered saline | Gibco | 14190144 | |
| Eosin Y solution | Sigma-Aldrich | E4009 | |
| Fetal Bovine Serum | Sigma-Aldrich | F9665 | |
| Formalin solution | Sigma-Aldrich | HT501128 | |
| GlutaMAX Supplement | Thermo Fisher Scientific | 35050061 | |
| HEK-293 | American Type Culture Collecti | ATCC CRL-1573 | |
| Hematoxylin solution | Sigma-Aldrich | 1051750500 | |
| Human primary glioma stem-like cells (GBM62) | Provided by Dr. E. A. Chiocca (Brigham and Women’s Hospital, Boston, MA) | ||
| Human primary glioma stem-like cells (MGG4) | Provided by Dr. Hiroaki Wakimoto (Massachusetts General Hospital, Boston, MA) | ||
| Lentiviral vector pCDH-CMV-MCS-EF1-copGFP | System Biosciences | CD511B-1 | |
| Lipofectamine 2000 | Thermo Fisher Scientific | 11668019 | |
| Microcentrifuge refrigerated | Eppendorf | model no. 5424 R, cat. no.5404000138 | |
| Mounting medium | Thermo Fisher Scientific | 4112APG | |
| Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes | Thermo Fisher Scientific | 3117-0380PK | |
| NanoDrop | Thermo Fisher Scientific | 2000c | |
| Neural Progenitor cells (NPC) | Provided by Dr. Jakub Godlewski (Brigham and Women’s Hospital, Boston, MA) | ||
| Neurobasal Medium | Thermo Fisher Scientific | 21103049 | |
| Nikon eclipse Ti motorized fluorescent microscope system | Nikon, Japan | 14314 | |
| Opti-MEM | Thermo Fisher Scientific | 31985088 | |
| PCR tubes | Sigma-Aldrich | CLS6571-960EA | |
| Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | |
| Petri-Dishes 94/16 | Greiner Bio-One | 632180 | |
| Poly-D-Lysine | Sigma- Aldrich | P4707 | |
| Recombinant Human EGF | PeproTech | AF-100-15 | |
| Recombinant Human FGF-basic | PeproTech | AF-100-18B | |
| Retinoic acid | Gibco | 12587-010 | |
| RNA Miniprep Kit | Direct-zol | R2050 | |
| S1000 Thermal Cycler | Bio-Rad | 1852196 | |
| Small Animal Image-Guided Micro Irradiator | Xstrahal Life Sciences, UK | Core facility (Dana-Farber Cancer Institute, Boston, MA) | |
| Sorvall WX+ Ultracentrifuge | Thermo Fisher Scientific | 75000100 | |
| StemPro Accutase | Thermo Fisher Scientific | A1110501 | |
| StepOne Real-Time PCR System | Applied Biosystems | 4376357 | |
| SterilGARD biosafety cabinet | The Baker Company | SG403A-HE | |
| Sucrose | Sigma-Aldrich | S9378 | |
| T98-G | American Type Culture Collecti | ATCC CRL-1690 | |
| TaqMan MicroRNA Reverse Transcription Kit | Thermo Fisher Scientific | 4366596 | |
| TaqMan Universal PCR Master Mix | Thermo Fisher Scientific | 4324018 | |
| Temozolomide | Tocris Bioscience | 2706 | |
| Tissue-Tek optimum cutting temperature | Fisher Scientific | NC9636948 | |
| TRIzol Reagent | Thermo Fisher Scientific | 15596026 | Lysis reagent |
| U251-MG | American Type Culture Collecti | ATCC HTB-17 | |
| U87-MG | American Type Culture Collecti | ATCC HTB-14 | |
| ViraPower Lentivector Expression system | Thermo Fisher Scientific | K4970-00 | |
| Water, HPLC grade | Fisher | W54 | |
| Xylene | Sigma-Aldrich | 534056 |
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Citar este artículo
Bhaskaran, V., Peruzzi, P. Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy. J. Vis. Exp. (152), e60215, doi:10.3791/60215 (2019).