We thank Anthony K. L. Leung of the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University for his technical help with designing the miR-181-sponge construct. We also thank Polina Sysa-Shah and Kathleen Gabrielson of the Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions for their technical assistance by the in vivo imaging of the miRNA-Sponge delivery.
This work was supported by grants from the NIH, HL39752 (to Charles Steenbergen) and by a Scientist Development Grant from the American Heart Association 14SDG18890049 (to Samarjit Das). The rat cardio-specific promoter was generously provided by Jeffery D. Molkentin at the Cincinnati Children&#39;s Hospital.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Johns Hopkins University.
1. Sponge Design
<ol>
	<li>MicroRNA binding 3&#8217;UTR 
	Note: A miRNA functions through specific base pairing interactions with partially complementary sites in the 3&#8217; untranslated region (UTR) of its target mRNAs (for a comprehensive review, see Bartel)15.
	<ol>
		<li>Design the inhibitors of miRNA expression as oligo...

<ol>
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This article described the design and synthesis of a miRNA-sponge and demonstrated how the tissue-specific expression of the sponge is a powerful tool to inhibit tissue-specific miRNA family expression.
We have demonstrated that a miR-181 family targeting sponge can be cloned into an expression plasmid with a cardiac-specific promoter. The plasmid can be efficiently packaged into a nanovector particle for delivery both in vitro and in vivo using electroporation or a tail vein...

Over the last two decades, there have been numerous studies that have pointed to the significant role of miRNAs in human disease. Findings from a large body of literature demonstrate the undeniable importance of miRNAs in the pathophysiology of diseases, such as cancer1 and cardiovascular disease2,3,4,5. For example, miR-21 is upregulated in many cancers, resulting in an increased cell cycle and cell proliferation6. In hepatitis C infections, miR-122 plays an important role in the replication of the virus7, and it has been shown that the inhibition of miR-122 decreases the viral load8. In cardiac hypertrophy, miR-212/132 is upregulated in the heart and is involved in the pathological phenotype9. The obvious importance of the downregulation or functional inhibition of an upregulated miRNA suggests opportunities for therapeutically exploiting the miRNA biology in almost all diseases.
The four miR-181 family members, miR-181a/b/c/d, are found in three genomic locations in the human genome. The intronic region of a non-coding RNA host gene (MIR181A1-HG) encodes the cluster of miR-181-a/b-1. The intronic region of the NR6A1 gene encodes the miR-181-a/b-2. The miR-181-c/d cluster is located in an uncharacterized transcript on chromosome 19. All the miR-181 family members share the same &#34;seed&#34; sequence and all four miR-181 family members can potentially regulate the same mRNA targets.
We3,4 and others10 have highlighted the importance of miR-181 family members during the end-stage heart failure. We have also recognized that a miR-181c upregulation occurs under pathological conditions associated with an increased risk of heart disease, such as type II diabetes, obesity, and aging3,4,5. It has been postulated that the overexpression of miR-181c causes oxidative stress which leads to a cardiac-dysfunction4.
Several groups have suggested that miRNA exist in mitochondria11,12,13,14, but we were the first to demonstrate that miR-181c is derived from the nuclear genome, processed, and subsequently translocated to the mitochondria in the RISC3. Furthermore, we have detected a low expression of miR-181a and miR-181b in the mitochondrial compartment of the heart5. Importantly, we have found that miR-181c represses the mt-COX1 mRNA expression, thereby demonstrating that miRNAs participate in the mitochondrial gene regulation and alter mitochondrial function3,4.
This article discusses the methodology required to design a miRNA-sponge to knock down the entire miR-181 family in cardiomyocytes. Moreover, we outline a protocol for the in vivo application of the miR-181-sponge.

<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) 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 the stably transfected pEGFP-miR-181-sponge-expressing H9c2 cells (from step 4.2), the expression of the entire miR-181 family (miR-181a, miR-181b, miR-181c, and miR-181d) was moderately decreased relative to pEGFP-scrambled-expressing H9c2 cells. MiR-181-sponge serves as a competitive inhibitor of the entire miR-181 family, so we anticipated that the expression of the miR-181c mitochondrial target gene, mt-COX1, would increase. Western blot data suggest that the mt-COX1 expression was...

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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.

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 ...

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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7.3K 조회수.  University of Toronto. 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 s...