Name: in vivo nanovector delivery of a heart-specific microrna-sponge
Uploaded: 2018-06-15T15:00:00
Description: Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

Agradecemos a Anthony K. L. Leung del Departamento de Bioquímica y Biología Molecular, Bloomberg School of Public Health, Universidad de Johns Hopkins para su técnico ayudar con el diseño de la construcción de la miR-181-esponja. También agradecemos a Polina Sysa-Shah y Kathleen Gabrielson de Departamento de Molecular y Biopatología comparada, Johns Hopkins Medical instituciones para su asistencia técnica de la proyección de imagen en vivo de la entrega de miRNA-esponja.
Este trabajo fue financiado por becas del NIH, HL39752 (a Charles Steenbergen) y por una beca de desarrollo científico de la 14SDG18890049 de la Asociación Americana del corazón (a Samarjit Das). El promotor de cardio-específica de la rata fue generosamente proporcionado por Jeffery D. Molkentin en Hospital infantil de Cincinnati.

Todos los procedimientos experimentales fueron aprobados por el cuidado de Animal institucional y uso Comité de la Universidad Johns Hopkins.
1. esponja diseño
<ol><li>
MicroARN enlace 3' UTR Nota: Funciones de miRNA A través de la base sincronización interacciones con sitios parcialmente complementarios en la región 3' no traducida (UTR) de sus mRNAs de destino (para una revisión exhaustiva, véase Bartel)15.<ol>
<li>Diseño de los inhibidor...

<ol>
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Este artículo describe el diseño y síntesis de una esponja de miRNA y demostró cómo la expresión específica de tejido de la esponja es una poderosa herramienta para inhibir la expresión familia miRNA de tejidos específicos.
Hemos demostrado que una familia miR-181 a esponja puede ser clonada en un plásmido de expresión con un promotor específico cardiaco. El plásmido puede empacarse eficientemente en una partícula nanovector para la entrega en vitro y en vivo us...

En las últimas dos décadas, ha habido numerosos estudios que han señalado el importante papel de miRNAs en enfermedad humana. Resultados de un gran cuerpo de literatura demuestran la indiscutible importancia de miRNAs en la patofisiología de enfermedades como el cáncer1 y las enfermedades cardiovasculares2,3,4,5. Por ejemplo, miR-21 es upregulated en muchos cánceres, lo que resulta en un mayor ciclo celular y la célula proliferación6. En las infecciones de hepatitis C, miR-122 desempeña un papel importante en la replicación de los virus7, y se ha demostrado que la inhibición de la miR-122 disminuye la carga viral8. En la hipertrofia cardiaca, miR-212/132 es upregulated en el corazón y está implicado en el fenotipo patológico9. La importancia obvia de la desregulación o la inhibición funcional de un miRNA alza sugiere oportunidades para explotar terapéuticamente la biología miRNA en casi todas las enfermedades.
Los cuatro miembros de la familia del miR-181, miR-181a/b/c/d, se encuentran en tres lugares genómicos en el genoma humano. La región intronic de una codificación no host gene del ARN (MIR181A1-HG) codifica el conjunto de miR-181-a/b-1. La región intronic del NR6A1 gen codifica el miR-181-a/b-2. El cluster de miR-181-c/d se encuentra en una transcripción desacostumbrada en el cromosoma 19. Todos los miR-181 miembros de la familia comparten la misma secuencia de "semilla" y los cuatro miembros de familia miR-181 potencialmente pueden regular los mismo objetivos de mRNA.
Somos3,4 y otros10 han destacado la importancia de miR-181 miembros de la familia durante la insuficiencia de la fase final. También hemos reconocido que un upregulation de miR - 181c ocurre bajo condiciones patológicas asociadas con un mayor riesgo de enfermedades del corazón, como el tipo de diabetes II, la obesidad y el envejecimiento3,4,5. Se ha postulado que la sobreexpresión de miR - 181c causa estrés oxidativo que conduce a una disfunción cardíaca4.
Varios grupos han sugerido que miRNA existe en mitocondrias11,12,13,14, pero fuimos los primeros en demostrar que miR - 181 c se deriva del genoma nuclear, procesado, y Posteriormente se translocan a la mitocondria en el RISC3. Además, hemos detectado una baja expresión de miR-181a y miR-181b en el compartimiento mitocondrial del corazón5. Lo importante, hemos encontrado que miR - 181c reprime la expresión de mRNA de COX1 mt, lo que demuestra que los miRNAs participan en la regulación del gene mitocondrial y altera la función mitocondrial3,4.
Este artículo aborda la metodología necesaria para diseñar una esponja miRNA derribar a toda la familia miR-181 en los cardiomiocitos. Por otra parte, describiremos un protocolo para el uso en vivo de la miR-181-esponja.

<table>
	<tbody>
		<tr>
			<td>pEGFP-C1 vector</td>
			<td>Addgene</td>
			<td>6084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>In-fusion</td>
			<td>Clontech</td>
			<td>121416</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Miniprep</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>miR-181-sponge synthesis</td>
			<td>Introgen GeneArt</td>
			<td>custome made</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td>custome</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI enzymes</td>
			<td>New Endland Biolabs</td>
			<td>R0101S</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI enzymes</td>
			<td>New Endland Biolabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid DNA Ligation Kit</td>
			<td>Sigma-Aldrich</td>
			<td>11635379001</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 cells</td>
			<td>ATCC</td>
			<td>CRL-1446</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082139</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector Kits for H9c2 (2-1)</td>
			<td>Lonza</td>
			<td>VCA-1005</td>
			<td></td>
		</tr>
		<tr>
			<td>G418, Geneticin</td>
			<td>Thermo Fisher Scientific</td>
			<td>11811023</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria II Flow cytometer</td>
			<td>BD Bioscience</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 450 sonifier</td>
			<td>Marshall Scientific</td>
			<td>EDP 100-214-239</td>
			<td></td>
		</tr>
		<tr>
			<td>The Xenogen IVIS Spectrum optical imaging device</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MTCO1 antibody</td>
			<td>Abcam</td>
			<td>ab14705</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab7291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequoia C256 ultrasound system</td>
			<td>Siemens</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo nanovector delivery of a heart-specific microrna-sponge

MicroRNA (miRNA) es pequeño no codificante del RNA que inhibe la expresión postranscripcional ARN mensajero (ARNm). Enfermedades humanas, como el cáncer y las enfermedades cardiovasculares, se han demostrado para activar el tejido o célula-específica miRNA expresión asociada a progresión de la enfermedad. La inhibición de expresión de miRNA ofrece la posibilidad de una intervención terapéutica. Sin embargo, los enfoques tradicionales para inhibir miRNAs, empleando oligonucleótidos antagomir afectan funciones específicas miRNA suministro global. Adjunto, presentamos un protocolo para la inhibición de cardio-específica en vivo de la familia miR-181 en un modelo de rata. Una construcción de miRNA-esponja está diseñada para incluir secuencias repetidas enlace anti-miR-181 10. El promotor de la cardio-específico α-MHC se clona en la columna vertebral de pEGFP para impulsar la expresión de miR-181 miRNA-esponja de cardio-específico. Para crear una célula estable línea expresando el miR-181-esponja, mioblastos H9c2 células transfected con el constructo α-MHC-EGFP-miR-181-sponge y ordenado por celular activado por fluorescencia (FACs) de clasificación en GFP positivas H9c2 las células que se cultivan con neomicina (G418). Después de un crecimiento estable en neomicina, poblaciones de células monoclonales son establecidas por FACs adicional y la clonación de la célula. Las células resultantes de H9c2-miR-181-esponja-GFP mioblastos exhiben una pérdida de función de miembros de la familia miR-181 según lo determinado por el aumento de la expresión de las proteínas diana de miR-181 y compararon H9c2 las células con una esponja no funcional de scramble. Además, desarrollamos un nanovector para la entrega sistémica de la construcción de la miR-181-esponja secuestrantes nanopartículas liposomal con carga positiva y carga negativa miR-181-esponja plásmidos. En vivo la proyección de imagen de GFP revela que múltiples inyecciones de vena de la cola de un nanovector durante un período de tres semanas son capaces de promover una expresión significativa de la miR-181-esponja de forma cardio-específico. Lo importante, se observa una pérdida de función de miR-181 en el tejido del corazón pero no en el riñón o el hígado. La esponja de miRNA es un método eficaz para inhibir la expresión del miRNA de tejidos específicos. Conducir a la expresión del miRNA-esponja de un promotor específico de tejido proporciona especificidad de la inhibición de miRNA, que puede limitarse a una específica órgano o tejido. Además, combinando tecnologías nanovector y miRNA-esponja permite una entrega efectiva y la inhibición de miRNA de tejidos específicos en vivo.

En las estable transfected pEGFP-miR-181-esponja-expresando H9c2 células (del paso 4.2), la expresión de miR-181 toda la familia (miR-181a, 181b miR, miR - 181c y miR - 181d) disminuyó moderadamente en comparación con células H9c2 pEGFP-revuelto-expresión. MiR-181-esponja sirve como un inhibidor competitivo de toda la familia miR-181, por lo que anticipó que la expresión del miR - 181c mitocondrial objetivo gene, mt-COX1, aumentaría. Mancha blanca /negra occidental los datos sugi...

Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

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

En Vivo Nanovector entrega de una esponja de microARN específicos de corazón

Inhibición de tejidos específicos microRNA es una tecnología que está poco desarrollada en el campo de microARN. Adjunto, describimos un protocolo para inhibir con éxito la familia de microRNA miR-181 en mioblastos células del corazón. Nanovector tecnología se utiliza para entregar un microRNA esponja que demuestra importantes en vivo cardio-específica miR-181 familia inhibición.

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

So for the last couple of decades, the importance of microRNA has been recognized in human disease conditions, but the problem is that, therapy-wise, it did not go to the primetime. One of the problem is, one microRNA can have different targets in different tissues. So one microRNA can be protected in one organ but detrimental to the others.So this method can help to eliminate tissue-specific microRNA knockdown strategy. The main advantage of this technology is we will use a microRNA sponge delivery and can be performed a tissue-specific manner. Begin this procedure with sponge design as described in the text protocol.To remove the CMV promoter from the commercially-available EGFR plasmid, first perform a digestion reaction. Following digestion, add five times the reaction volume of column binding buffer. Purify the digested linearized plasmid with a DNA purification column as described by the manufacturer.Elute with 25 microliters of elution buffer added carefully to the center column. Gel-purify the digested plasmid on a 0.5%agarose gel by first adding five microliters of loading buffer to the eluded plasmid. Load the entire sample into one well on a 0.5%agarose gel.Include a separate lane with 500 nanograms of uncut vector. Run it for 30 to 40 minutes, until an adequate separation of cut and uncut plasmids is achieved. After cloning the cardio-specific promoter alpha-myosin heavy chain as subscribed in the text protocol, add five times the reaction volume of the column binding buffer to the PCR reaction and purify it with a DNA purification column as described by the manufacturer.Elute the mixture with 25 microliters of elution buffer added carefully to the center of the column. Add five microliters of loading buffer to the eluded plasmid and load the entire sample into one well of a 1%agarose gel. Run the gel for 30 to 40 minutes before excising the PCR product as before.Ligate the alpha-myocin heavy chain promoter with the EGFP plasmid at 50 degrees Celsius for 15 minutes before cooling the reaction on ice. Perform a bacterial transformation by transfecting 50 microliters of competent cells with 2.5 microliters of infusion ligation mix. Rest the Eppendorf tube on ice for 20 minutes.Then, incubate the reaction in a 42 degree Celsius water bath for 40 seconds and place the tube on ice for two minutes. Following the transformation, add 350 microliters of SOC media and grow the cells at 37 degrees Celsius for 45 minutes. Plate 200 microliters of the outgrowth solution on LB plates with Kanamycin.Perform a colony PCR to identify Kanamycin-resistant cells, which contain the alpha-myosin heavy chain promoter ligation. Ligate the microRNA 181 sponge into the alpha-myosin heavy chain expressed EGFP vector by setting up a 21 microliter ligation reaction as detailed in the text protocol. Perform the ligation at room temperature for five minutes and then place it on ice.Transform 50 microliters of XL1-Blue competent cells with two microliters of ligation mix as before. Perform a colony PCR to identify Kanamycin-resistant bacteria, which contain the correct plasmid cloned with the first alpha-myacin heavy chain followed by the microRNA 181 sponge construct sequence. Design screening primers to PCR across the ligation junction.Expand the PCR positive clones in LB Kanamycin broth and in plasmids purified using standard mini-prep plasmid purification protocols. Perform DNA sequencing to verify that the plasmid is expressing the desired DNA sequences. Using an electroporator, transfect rat myoblasts with either a scramble sponge as a control, or the plasmid cloned with the first alpha-myocin heavy chain, followed by the microRNA 181 sponge construct.Briefly, transfect 0.4 million cells with two microgams of plasmid DNA and 100 microliters of a solution compatible with the electroporation device. Use an appropriate program to transfect the H9C2 cells. Plate only the GFP-expressed cells into a six-well plate with an addition of neomycin to the complete growth media.After seeding five to 10 cells in each well of a 96-well plate, followed by flow sorting, expand the monoclonal cells from the 96-well plate to 24-well plates over several passages. Prepare the liposomal nanoparticles by dissolving the components listed in the text protocol in a mixture of chloroform and methanol in a glass vial. Prepare the mixture by adding 5%glucose to the vacuum-dried lipid film before leaving it overnight to allow this mixture to hydrate the film.Gently rock the vial for two to three minutes at room temperature in order to produce multilamellar vesicles. Then, incubate the vesicles with occasional shaking in a 45 degree Celsius incubator. Sonicate the multilamellar vesicles to prepare small unilamellar vesicles in an ice bath for three to four minutes, until clarity can be observed.Add a 100%duty cycle and 25 watts of output power. Mix either a scramble sequence or a microRNA 181 sponge sequence cloned into the alpha-myocin heavy chain expressed EGFP plasmid with liposome on a one-to-three charge ratio basis to form the nanovector. The results of optimized three-week treatment protocols with six intravenous injections through the tail vein are shown here.The yellow colorization in the epifluorescence image demonstrates expression of the EGFP vector. The maximum intensity of yellow colorization is observed at the week three time point. The alpha-myosin heavy chain promoter sequence in the EGFP vector selectively expresses either microRNA 181 sponge or scrambled sequence in the heart at Day 21 as revealed by the yellow fluorescence.The Western blot and band densitometry showed a significant increase in the mt-COX1 expression in the microRNA 181 sponge nanovector injected rats as compared to the scramble nanovector groups, with alpha-Tubulin as the normalization control. One of the major challenges in this particular protocol is to keep the orientation of the sequencing, you need directional. Once a bacterial preparation is made, with a proper orientation of the microRNA sponge, this technique can be done with a two or three minute tail vein injection time.While attempting this procedure, it is important to sequence your bacterial colony multiple times, multiple batches, to make sure the microRNA response sequence is in the correct order. After watching this video, you should have a good understanding of how to design a microRNA sponge to knock down the expression of any microRNA family and prepare an in vivo application of microRNA sponge. Following this procedure, other methods, such as in vivo antagomir treatment can be performed to answer additional questions like whether a specific microRNA can be knocked down in all the organ at the same time.After its development, this particular technology paved the way for researcher in the field of microRNA to explore tissue-specific knockdown of a microRNA in the pathophysiology of multiple-disease conditions. Tissue-specific microRNA inhibition is currently an undeveloped technique in the microRNA field. The importance of mat-down regulation of functional inhibition of up-regulated microRNAs in human disease suggest exploring microRNA biology to create a cellular vulnerability for therapeutic intervention.

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Biology

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

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