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&#8217;Hospital de la Santa Creu i Sant Pau) Barcelona (Spain) for gifting the CMV-Firefly luc- IRES-EGFP and Cal62-Luc cells, respectively.&#160;Funding:&#160; SAF2016-75531-R (MICIU), Fondo Europeo de Desarrollo Regional FEDER, B2017/BMD-3724 (Comunidad de Madrid), and GCB14142311CRES (Fundaci&#243;n Espa&#241;ola contra el C&#225;ncer, AECC).

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 Cient&#237;ficas (CSIC, Spain).
1. Flank inoculation of cells and intratumoral antagomiR treatment
<ol>
	<li>Cell preparation
	<ol>
		<li>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 by sorting the GFP-positive cells with flow cytometry.</li>
		<li>Grow the cells in Dulbecco&#180;s modified Eagle&#180;s medium (DMEM) supplemented with penicillin, streptomycin and 10% fetal bovine serum (FBS) at 37 &#176;C and 5% CO2.</li>
		<li>Suspend 1 x 106 cells in 50 &#956;L of phosphate buffered saline (PBS) at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cell inoculation into the flanks of mice 
	<ol>
		<li>Mix the cells with the same amount of basement membrane matrix. For example, add 1 x 106 cells in 50 &#956;L of PBS to 50 &#956;L of basement membrane matrix (see Table of Materials) and mix gently. 
		NOTE:</li>
		<li>Inject 100 &#956;L of the sample (cells in PBS + basement membrane matrix) subcutaneously into the left flank of 6-week-old immunodeficient BALB/c nu/nu mice using a 1 mL insulin syringe with a 27G 1/2&#39;&#39; (0.4x13 mm) needle.</li>
	</ol>
	</li>
	<li>Intratumoral antagomiR treatment. 
	<ol>
		<li>Suspend the antagomiR (see Table of Materials) or the negative control in 500 &#181;L of RNase-free distilled water.</li>
		<li>Prior to the injection, prepare 2 nmol of the antagomiR or the control together with an in vivo delivery reagent (see Table of Materials) for each injection.
		<ol>
			<li>First mix the antagomiR solution (see Table of Materials) and the complexation buffer (see Table of Materials) in a 1:1 ratio. For example, add 80 &#956;L of miRNA-inhibitor solution (16 nmol) to 80 &#956;L of complexation buffer (included in the in vivo delivery reagent kit, see Table of Materials).</li>
			<li>Bring the in vivo delivery reagent to room temperature. Add 160 &#956;L to a 1.5-mL tube and immediately add 160 &#956;L of diluted antagomiR solution. Return remaining reagent to -20 &#176;C. If necessary, store the reagent at 4 &#176;C for up to one week after thawing.</li>
			<li>Vortex immediately (10 s) to ensure complexation of the in vivo delivery reagent-antagomiR.</li>
			<li>Incubate the in vivo delivery reagent-antagomiR mixture for 30 min at 50 &#176;C. Centrifuge the tube briefly to recover the sample.</li>
			<li>Dilute the complex 6-fold by adding 1360 &#181;L of PBS pH 7.4 and mix well.</li>
			<li>Proceed with in vivo delivery of the reagent-antagomiR complex (8 mice/condition), or store the complex at 4 &#176;C for up to one week prior to injection. Inject 200 &#956;L intratumorally into each tumor (2 nmol of antagomiR). 
			NOTE: The volume and quantity of the antagomiR is independent of the tumor volume.</li>
		</ol>
		</li>
		<li>Perform the treatment 3 times each week (Monday, Wednesday and Friday) for 2 weeks.</li>
	</ol>
	</li>
	<li>Analysis of tumor growth
	<ol>
		<li>Inject 50 &#956;L of a 40 mg/mL solution of D-luciferin substrate (see Table of Materials) subcutaneously at each time point with a 1 mL syringe with a 27G 1/2&#39;&#39; (0.4 mm x 13 mm) needle.
		<ol>
			<li>At 8 min post-injection, anesthetize the mice using 3% isoflurane mixed with oxygen. Assess the level of anesthesia by pedal reflex (firm toe pinch) and adjust anesthetic delivery as appropriate to maintain surgical plane.</li>
			<li>Apply ophthalmic ointment to both eyes to prevent desiccation.</li>
		</ol>
		</li>
		<li>Image the bioluminescent signal with in vivo imaging software (see Table of Materials). 
		NOTE: Calipers can also be used to measure the tumor volume and the tumor growth.</li>
		<li>Once the bioluminescence signals are obtained, analyze the tumoral growth comparing both treatments and determine the significance by using a t-test. To analyze the within-group variance use the SEM.</li>
	</ol>
	</li>
	<li>Tumor excision
	<ol>
		<li>Seven days after the end of the antagomiR treatment, sacrifice the mice, excise the tumor and extract protein and/or RNA for future analysis. Alternatively, section the tumors and fix them for immunohistochemistry.</li>
	</ol>
	</li>
</ol>
2. Thyroid orthotopic inoculation of cells and systemic antagomiR treatment
<ol>
	<li>Cell preparation
	<ol>
		<li>Engineer a Cal62 human thyroid cancer cell line to overexpress a transgenic construct that constitutively expresses GFP and luciferase. Select the transgenic cells by antibiotic resistance or by sorting the GFP-positive cells with flow cytometry.</li>
		<li>Grow these cells in DMEM supplemented with antibiotics (penicillin and streptomycin) and 10% FBS at 37 &#176;C and 5% CO2.</li>
		<li>Suspend 1 x 105 cells in 5 &#956;L of PBS.</li>
	</ol>
	</li>
	<li>Cell inoculation into the thyroid gland of mice
	<ol>
		<li>Inject 100 &#956;L of analgesic (buprenorphine) and 100 &#956;L of antibiotic (cephalosporin) subcutaneously into 7-week-old BALB/c nu/nu mice.&#160;Disinfect the injection site with alcohol.</li>
		<li>Inject 5 &#956;L of the cell solution into the thyroid gland of the mice.
		<ol>
			<li>Anesthetize the mouse using 3% isoflurane mixed with oxygen and place it under a stereomicroscope in a sterile flow cabinet.&#160;&#160;Assess the level of anesthesia by pedal reflex (firm toe pinch) and adjust anesthetic delivery as appropriate to maintain surgical plane.</li>
			<li>Apply ophthalmic ointment to both eyes to prevent desiccation.&#160;</li>
			<li>For each mouse, disinfect the neck with iodopovidone and make an incision of approximate 2 cm in the skin with scissors. Once open, expose the neck by displacing the salivary glands.</li>
			<li>Dissect the strap muscles using dissection forceps and/or scissors to expose the trachea and the thyroid gland. Using a 10 &#956;L microliter syringe inject 5 &#956;L of the cell solution into the right thyroid lobule, located at the side of the cricoid cartilage.</li>
			<li>Reposition the salivary glands and suture the incision with silk using braided, coated, non-absorbable sutures.</li>
			<li>Add iodopovidone to the wound area and place the mouse on a thermic blanket while it recovers from the anesthesia.</li>
		</ol>
		</li>
		<li>The following day post-surgery, inject subcutaneously analgesic (buprenorphine) and add 3 mL of liquid ibuprofen (40 mg/mL) into 250 mL of the drinking water for 1 week.</li>
	</ol>
	</li>
	<li>Systemic antagomiR treatment 
	NOTE: Perform this step 2-3 weeks after the cell injection, when the bioluminescent signal is detectable.
	<ol>
		<li>Suspend the antagomiR or the negative control in distilled RNase-free water.</li>
		<li>Prior to the injection, prepare 7 nmol of the antagomiR or the control together with the in vivo delivery reagent for each injection.
		<ol>
			<li>For the antagomiR and the in vivo delivery reagent solution preparation see step 1.3 but add 7 nmol of the miRNA-inhibitor per mouse.</li>
		</ol>
		</li>
		<li>Administer the solution intravenously by retro-orbital injection of the venous sinus of the mouse. Use isoflurane to induce anesthesia. 
		NOTE: Assess the level of anesthesia by pedal reflex (firm toe pinch).</li>
		<li>Treat the mice 3 times a week (Monday, Wednesday and Friday) for 2 weeks.</li>
	</ol>
	</li>
	<li>Analysis of tumor growth
	<ol>
		<li>Determine the tumor bioluminescent signal twice weekly to calculate tumor growth.
		<ol>
			<li>Inject 50 &#956;L of a 40 mg/mL solution of D-luciferin substrate at each time point via subcutaneous injection.</li>
			<li>At 8 min post-injection, anesthetize the mice using 3% isoflurane mixed with oxygen and image the bioluminescent signal with in vivo imaging software.</li>
		</ol>
		</li>
		<li>Once the bioluminescence signals are obtained, analyze the tumoral growth comparing both treatments and determine the significance by using a t-test. To analyze the within-group variance use the SEM.</li>
	</ol>
	</li>
	<li>Tumor excision
	<ol>
		<li>Seven days after the end of the antagomiR treatment period, sacrifice the mice, excise the tumor and extract protein and/or RNA for future analysis. Alternatively, section the tumors and fix them for immunohistochemistry.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lim, H., Devesa, S. S., Sosa, J. A., Check, D., Kitahara, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+Thyroid+Cancer+Incidence+and+Mortality+in+the+United+States.">Trends in Thyroid Cancer Incidence and Mortality in the United States.</a> Journal of the American Medical Association. 317 (13), 1338-1348 (2017).</li><li>Landa, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+transcriptomic+hallmarks+of+poorly+differentiated+and+anaplastic+thyroid+cancers.">Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers.</a> The Journal of Clinical Investigation. 126 (3), 1052-1066 (2016).</li><li>Gregory, R. I., Shiekhattar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+biogenesis+and+cancer.">MicroRNA biogenesis and cancer.</a> Cancer Research. 65 (9), 3509-3512 (2005).</li><li>Lin, S., Gregory, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+biogenesis+pathways+in+cancer.">MicroRNA biogenesis pathways in cancer.</a> Nature Reviews Cancer. 15 (6), 321-333 (2015).</li><li>Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+oncogenes.">MicroRNAs as oncogenes.</a> Current Opinion In Genetics &amp; Development. 16 (1), 4-9 (2006).</li><li>Cancer Genome Atlas Reserch Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+genomic+characterization+of+papillary+thyroid+carcinoma.">Integrated genomic characterization of papillary thyroid carcinoma.</a> Cell. 159 (3), 676-690 (2014).</li><li>Li, Z., Rana, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+microRNAs:+current+status+and+future+challenges.">Therapeutic targeting of microRNAs: current status and future challenges.</a> Nature Reviews. Drug Discovery. 13 (8), 622-638 (2014).</li><li>Pallante, P., Battista, S., Pierantoni, G. M., Fusco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulation+of+microRNA+expression+in+thyroid+neoplasias.">Deregulation of microRNA expression in thyroid neoplasias.</a> Nature Reviews. Endocrinology. 10 (2), 88-101 (2014).</li><li>Fuziwara, C. S., Kimura, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+thyroid+development,+function+and+tumorigenesis.">MicroRNAs in thyroid development, function and tumorigenesis.</a> Molecular and Cellular Endocrinology. 456, 44-50 (2017).</li><li>Fuziwara, C. S., Kimura, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+Deregulation+in+Anaplastic+Thyroid+Cancer+Biology.">MicroRNA Deregulation in Anaplastic Thyroid Cancer Biology.</a> International Journal of Endocrinology. 2014, 743450 (2014).</li><li>Riesco-Eizaguirre, G., Santisteban, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+Tumours:+Advances+in+the+molecular+pathogenesis+of+thyroid+cancer:+lessons+from+the+cancer+genome.">Endocrine Tumours: Advances in the molecular pathogenesis of thyroid cancer: lessons from the cancer genome.</a> European Journal of Endocrinology. 175 (5), R203-R217 (2016).</li><li>Riesco-Eizaguirre, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+miR-146b-3p/PAX8/NIS+Regulatory+Circuit+Modulates+the+Differentiation+Phenotype+and+Function+of+Thyroid+Cells+during+Carcinogenesis.">The miR-146b-3p/PAX8/NIS Regulatory Circuit Modulates the Differentiation Phenotype and Function of Thyroid Cells during Carcinogenesis.</a> Cancer Research. 75 (19), 4119-4130 (2015).</li><li>Lima, C. R., Geraldo, M. V., Fuziwara, C. S., Kimura, E. T., Santos, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNA-146b-5p+upregulates+migration+and+invasion+of+different+Papillary+Thyroid+Carcinoma+cells.">MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells.</a> BMC Cancer. 16, 108 (2016).</li><li>Lee, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-222+and+microRNA-146b+are+tissue+and+circulating+biomarkers+of+recurrent+papillary+thyroid+cancer.">MicroRNA-222 and microRNA-146b are tissue and circulating biomarkers of recurrent papillary thyroid cancer.</a> Cancer. 119 (24), 4358-4365 (2013).</li><li>Deng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-146b-5p+promotes+metastasis+and+induces+epithelial-mesenchymal+transition+in+thyroid+cancer+by+targeting+ZNRF3.+Cellular+Physiology+and+Biochemistry.">MiR-146b-5p promotes metastasis and induces epithelial-mesenchymal transition in thyroid cancer by targeting ZNRF3. Cellular Physiology and Biochemistry.</a> International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 35 (1), 71-82 (2015).</li><li>Ramirez-Moya, J., Wert-Lamas, L., Santisteban, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-146b+promotes+PI3K/AKT+pathway+hyperactivation+and+thyroid+cancer+progression+by+targeting+PTEN.">MicroRNA-146b promotes PI3K/AKT pathway hyperactivation and thyroid cancer progression by targeting PTEN.</a> Oncogene. , (2018).</li><li>Ramirez-Moya, J., Wert-Lamas, L., Riesco Eizaguirre, G., Santisteban, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+microRNA+processing+by+DICER1+downregulation+endows+thyroid+cancer+with+increased+aggressiveness.">Impaired microRNA processing by DICER1 downregulation endows thyroid cancer with increased aggressiveness.</a> Oncogene. , (2019).</li><li>Xing, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+and+mechanisms+of+thyroid+cancer.">Molecular pathogenesis and mechanisms of thyroid cancer.</a> Nature Reviews. Cancer. 13 (3), 184-199 (2013).</li></ol>

The authors have nothing to disclose.

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 ...

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&#39; untranslational region (3&#8217;UTR) of target messenger RNAs (mRNAs), triggering mRNA degradation or translational repression3,4. There is increasing evidence demonstrating that the deregulation of microRNA expression is a hallmark of cancer, as these molecules modulate proliferative signaling, migration, invasion and metastasis, and can provide resistance to apoptosis5,6. In recent years, many studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis as well as therapeutic targets7, providing a new dimension to cancer evaluation and treatment.
miRNAs have taken center stage in human molecular oncology as key drivers of human thyroid neoplasms8,9,10,11,12. Among the miRNAs up-regulated, miR-146b is highly overexpressed in Papillary Thyroid Carcinoma (PTC) tumors and was shown to significantly increase cell proliferation, and to be associated with aggressiveness and dismal prognosis6,12,13,14,15. Furthermore, miR-146b regulates several thyroid genes involved in differentiation12, and also important tumor suppressor genes such as PTEN16 and DICER117. Despite their importance in cancer biology, miRNA-based cancer therapy is still in its early stages, and very few studies have addressed thyroid cancer - the most frequent of the endocrine tumors18. Here we describe a protocol using two different mouse models with human-derived tumors, in which the administration of a synthetic miRNA-inhibitor (antagomiR) that specifically inhibits a cellular miRNA can block tumor growth. We first used a common xenograft model, and the local intratumor administration of an antagomiR decreased tumor growth measured as a reduction in tumor bioluminescence16. Because the establishment of robust mouse models mimicking human tumor progression is essential to develop unique therapeutic approaches, orthotopic implantation of primary human tumors is a more valuable platform for clinical validation of new drugs than subcutaneous implantation models. Thus, in order to better assess the therapeutic potential of the antagomiR, we used an orthotopic mouse model with systemic delivery in the blood stream, obtaining the same results.

<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>&lt;em&gt;In vivo&lt;/em&gt; delivery reagent: Invivofectamine 3.0 Reagent</td><td>Thermo Fisher</td><td>IVF3005</td><td></td></tr><tr><td>&lt;em&gt;In vivo&lt;/em&gt; 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

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.

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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In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

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.

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

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Research

JoVE Journal

Cancer Research

7.2K Views.  Instituto de Investigaciones Biomédicas, CSIC-UAM. We voeren de eerste therapie uit, gebaseerd op een microRNA-remmer in een schildklierkankermodel. Deze ontwikkelende therapieën tonen belofte voor de behandeling van deze ziekte. Dit type orthotopisch model, met behulp van een systemische route van de behandeling levering, vergemakkelijkt de validatie van een nieuwe microRNA-gebaseerde geneesmiddelen.Hoewel we gebruik maken van een orthotopisch model voor het implanteren van menselijke schildklierkankercellen in de muis schildklieren,...

Video: In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Medicine

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Genetics

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Biology

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model

Source: Julia Ramirez-Moya, et al. <a href="https://www.jove.com/video/59322" target="_blank">In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.</a> J. Vis. Exp. (2019)
This video describes the procedure for orthotopic inoculation of thyroid cancer cells into the thyroid gland of mouse models, which may then serve as a valuable platform to assess carcinogenesis and discover potential treatment strategies.

Source: Julia Ramirez-Moya, et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)
This video describes the procedure ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

In vivo studies

Modelling Intratumoral Treatment with miRNA Inhibitors: A Method to Analyze the Effect of miRNA Inhibitors on Tumor Growth in Murine Model

Source: Ramirez-Moya, J. et al. <a href="https://www.jove.com/video/59322" target="_blank">In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.</a> J. Vis. Exp. (2019)
This video demonstrates the in vivo delivery of miRNA inhibitors as therapeutic agents against tumor growth in orthotopic mouse models. The intratumoral administration of synthetic miRNA inhibitors or antagomiRs allows them to bind and inhibit endogenous miRNAs, thereby causing mRNA upregulation and suppression of tumor growth.

Source: Ramirez-Moya, J. et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)
This video demonstrates the in vivo ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

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