方法文章

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

DOI：

10.3791/59322

⸱

2019年8月23日

 ,  ,  ,  , 
1Instituto de Investigaciones Biomédicas, CSIC-UAM, 2Ciberonc, Instituto de Salud Carlos III

本文内容

摘要

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This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

摘要

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MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

引言

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Thyroid cancer is an endocrine malignancy with an increasing incidence, although in general terms it has a good outcome1. Nevertheless, some patients develop aggressive forms of the disease that are untreatable and the molecular bases are poorly understood2.

miRNAs are 22-nucleotide-long non-coding RNAs that regulate gene expression in many tissues, typically by base-pair binding to the 3' untranslational region (3’UTR) of target messenger RNAs (mRNAs), triggering mRNA degradation or translational repression3,4. There is incr....

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方案

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Animal experimentation was performed in compliance with the European Community Law (86/609/EEC) and the Spanish law (R.D. 1201/2005), with approval of the Ethics Committee of the Consejo Superior de Investigaciones Científicas (CSIC, Spain).

1. Flank inoculation of cells and intratumoral antagomiR treatment

  1. Cell preparation
    1. Engineer a Cal62 human thyroid cancer cell line (KRASG12R and p53A161D mutations) to overexpress a transgenic construct that constitutively expresses GFP and luciferase (CMV-Firefly Luc-IRES-eGFP). Select the transgenic cells by antibiotic resistance or ....

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结果

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We used two different mice models to determine whether the neutralization of a miRNA could suppress tumor growth. Accordingly, human tumor thyroid Cal62-luc cells were subcutaneously injected into the flanks of nude mice to generate a xenograph model. After two weeks, tumors were established and could be measured with calipers. At that time point, mice were injected intratumorally with the miR-146b-inhibitor, or an appropriate control, and tumor volume was followed for a further two weeks (Figure 1A<.......

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讨论

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This paper describes a method for studying the in vivo function of a miRNA in order to better understand its role in tumor initiation and progression, and its potential as a therapeutic target in thyroid cancer. The tumor xenograft models here described are based on the use of cells that can be tracked by their bioluminescence signal, permitting the measurement of tumor growth in vivo under the influence of a treatment. In addition, we describe the use of a miRNA-based treatment for thyroid cancer, which is currently in .......

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披露

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The authors have nothing to disclose.

致谢

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We are grateful to Raquel Arocha Riesco for her assistance with the treatment and care of mice. We thank Dr. J. Blanco (Catalonian Institute for Advance Chemistry-CSIC) and Dr E. Mato (Institut de Reserca de l’Hospital de la Santa Creu i Sant Pau) Barcelona (Spain) for gifting the CMV-Firefly luc- IRES-EGFP and Cal62-Luc cells, respectively. Funding:  SAF2016-75531-R (MICIU), Fondo Europeo de Desarrollo Regional FEDER, B2017/BMD-3724 (Comunidad de Madrid), and GCB14142311CRES (Fundación Española contra el Cáncer, AECC).

....

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材料

本文使用的材料清单
姓名公司目录编号评论
AntagomiR：mirVana miRNA 抑制剂Thermo Fisher4464088体内就绪
基底膜基质：基质胶基底膜基质 高浓度Corning#354248
DICER 抗体Abcamab14601IHQ：1/100
体内 递送试剂：Invivofectamine 3.0 试剂Thermo FisherIVF3005
体内成像软件：IVIS-Lumina II 成像系统Caliper 生命科学
阴性对照：mirVana miRNA 抑制剂，阴性对照 #1Thermo Fisher4464077体内就绪
PCNA 抗体Abcamab92552WB：1/2,000
PTEN 抗体Santa Cruzsc-7974WB：1/1,000
XenoLight D-荧光素 - K+ 盐生物发光底物PerkinElmer122799在 PBS 中稀释

参考文献

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  1. Lim, H., Devesa, S. S., Sosa, J. A., Check, D., Kitahara, C. M. Trends in Thyroid Cancer Incidence and Mortality in the United States. Journal of the American Medical Association. 317 (13), 1338-1348 (2017).
  2. Landa, I., et al.

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标签

microRNA AntagomiR CAL 62 D

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