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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 α-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 α-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.

Introduction

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 "seed" 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.

Protocol

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Johns Hopkins University.

1. Sponge Design

  1. MicroRNA binding 3’UTR
    Note: A miRNA functions through specific base pairing interactions with partially complementary sites in the 3’ untranslated region (UTR) of its target mRNAs (for a comprehensive review, see Bartel)15.
    1. Design the inhibitors of miRNA expression as oligonucleotides that contain significant complementarity to the miRNA sequence. Sequester a miRNA in an oligonucleotide complex through extensive base pairing to block the miRNA function.
  2. Design of a miRNA sponge
    1. Design tandemly arrayed partially complementary miRNA sequences to include a bulge at the position normally cleaved by Argonaute 2. The bulge is positioned at nucleotides 9 - 12 of the mature miRNA sequence to prevent any RNA interference type cleavage and degradation of the sponge.
    2. If desired, design the decoy target sequence to bind to all miR-181 family members. Upon an examination and comparison of the miR-181 family miRNA sequences, find a common sequence at the 5’-side of the bulge containing 8 consecutive nucleotides, or find a sequence common to all 4 miR-181 family members on the 3’-side of the bulge of an additional 8 consecutive nucleotides. These 2 sequences represent the left and right half binding sites, respectively.
      Note: The decoy sequence is a tandem array of the left and right half binding sites separated by a GGA spacer. Link together the 5’-end sequence and the 3’-side sequence (described in step 1.2.2) with a GGA spacer designed to create the bulge when annealed to a miRNA (as described in step 1.2.1). Repeat this 1 miRNA binding site 10x in the final miR-sponge construct.
  3. Other miRNA sponge considerations
    Note: In order to clone the sponge sequence into the recipient expression vector, restriction nuclease recognition sites must be included at each end of the sequence. An in silico digestion analysis should be used to confirm the non-specific cleavage of the sponge sequence by the chosen restriction enzymes and other restriction enzymes used in downstream cloning applications.
    1. Add a double stop codon (TAA TAA) at the 5’-end of the synthetic 3’UTR, such that the miR-181-sponge sequence is not part of the coding sequence for an enhanced green fluorescent protein (EGFP).
  4. Synthesis of the sponge DNA for cloning
    1. Use a DNA synthesizer or employ a commercially available source to synthesize the final miRNA sponge sequence with restriction nuclease recognition sites in place for cloning. The sponge is shipped on a plasmid which can contain selectable markers for an amplification in bacteria. Further, the sponge can be amplified by a polymerase chain reaction (PCR) for cloning or simply dropped out the donor vector using the restriction sites described in step 1.3 (also, see step 3.1).

2. Cloning the Recipient Vector to Include a Tissue-specific Promoter

Note: Use the pEGFP-C1 vector as the recipient vector for the expression cassette of the miRNA sponge. The pEGFP-C1 vector contains a reading frame for an EGFP driven by a cytomegalovirus (CMV) promoter. The CMV promoter is constitutively active in mammalian cells. If a tissue-specific promoter is required, the CMV promoter can be removed by digestion of AseI and NheI enzymes (see step 2.1). The plasmid contains an extensive multiple cloning site (MCS) as well as kanamycin (Kan) and neomycin (G418) markers for a selection in bacteria and a stable expression in mammalian cells, respectively.

  1. Removing the CMV promoter from pEGFP-C1
    1. Make 50 μL of digestion reaction containing 1x commercially available digest buffer suggested by the manufacture of the restriction enzymes used, 5 μg of plasmid DNA (in water), and 5 μL each of AseI and NheI enzymes. Add all components to a microcentrifuge tube and gently mix them by flicking it. Spin the tube for 10 s in a microcentrifuge to collect the reaction at the bottom. Incubate the reaction in a 37 °C water bath for 15 min.
    2. Purify the digested linearized plasmid. Add 5x the reaction volume of column binding buffer (a proprietary mix of the appropriate binding buffer for the manufacture of the DNA purification columns used) and purify the reaction with a DNA purification column as described by the manufacturer. Elute with 25 μL of elution buffer (water) added carefully to the center of the column.
    3. Gel purify the digested plasmid on 0.5% agarose gel as follows: add 5 μL of loading buffer (50% glycerol-3x loading dye) to the eluted plasmid. Load the entire sample into 1 well on 0.5% agarose gel. Include a separate lane with 500 ng of uncut vector. Run it for 30 - 40 min until an adequate separation of cut and uncut plasmids is achieved.
    4. Purify the linearized plasmid as follows: visualize the DNA in the agarose gel on a UV light box. With a clean razor blade, carefully excise the band corresponding to the linear plasmid.
      Note: The linear plasmid should run lower than the uncut plasmid and will appear as a single band at approximately 4.2 kb. The excised CMV promoter will appear as a light band at approximately 500 nucleotides (nt).
    5. Extract the DNA from the gel slice using a commercially available kit and elute the DNA from the column with 25 μL of water.
  2. Cloning a heart-specific promoter into pEGFP-C1
    Note: The alpha-myosin heavy chain (α-MHC) promoter has been previously characterized and demonstrated to direct robust levels of cardiac-restricted expression as described in the following Molkentin, Jobe, and Markham16. The next sections describe how to amplify the promoter of the vector for cloning.
    1. Clone the α-MHC promoter between elements +420 to -2934 relative to the transcription start site into the p40 plasmid16 by first designing PCR primers that flank this region and then the plasmid as a template for PCR. Amplify the promoter region using PCR primers previously described5. Forward and reverse PCR primers contain sequences of overlap to the digested ends of the pEGFP-C1 (step 2.1) to allow In-Fusion directed cloning. Consult the In-Fusion manual for a comprehensive explanation of the primer design for In-Fusion cloning. Primer sequences are found in Table 1.
    2. Prepare a 50 μL PCR reaction, which contains 1 μM of each primer and 10 ng of template DNA. Add an appropriate amount of PCR enzyme and dNTPs depending on the manufacture of the PCR reagents chosen. Perform PCR on a standard DNA cycling block. Perform a 3 min 98 °C denaturing step, followed by 35 cycles of denaturing, annealing, and extension for 5, 30, and 120 s each, respectively. Include a final extension of 10 min at 72 °C.
    3. Clean up the PCR and gel extraction. Upon completion of the PCR cycle, add 5x 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 μL of elution buffer (water) added carefully to the center of the column.
      1. Add 5 μL of loading buffer [50% glycerol (3x) loading dye] to eluted plasmid. Load the entire sample into 1 well on 1% agarose gel. Run it for 30 - 40 min; visualize and excise the PCR product as described above (step 2.1.5).
    4. Ligate the α-MHC promoter with the pEGFP-C1 as follows: add the 10 μL ligation reaction containing 1x commercially available ligation buffer, 2 μL of purified PCR, and 1 μL of linearized vector. Perform the ligation at 50 °C for 15 min and then cool it on ice.
    5. Perform a bacterial transformation as follows: transfect 50 μL of Competent Cells with 2.5 μL of In-Fusion ligation mix by adding the components. Rest the Eppendorf tube on ice for 20 min, put it in a 42 °C water bath for 40 s, and then place the tube on ice for 2 min.
      1. Following the transformation, add 350 μL of SOC media and grow cells at 37 °C for 45 min. Plate 200 μL of outgrowth solution on LB plates with Kan. Perform a colony PCR to identify Kan resistant cells which contain the α-MHC promoter ligation.
        Note: Screening primers were designed to PCR across the ligation junction.
    6. Expand the PCR positive clones in LB-Kan broth and in plasmids purified using standard mini-prep plasmid purification protocols as described by the manufacturer.

3. Cloning the Sponge

  1. Digest the recipient vector
    1. Make the α-MHC-pEGFP plasmid linear for cloning with EcoRI and KpnI, and purify the gel as described above (step 2.1).
  2. PCR amplify and digest the sponge DNA
    1. Use the miR-181-sponge and corresponding 284-nt-long scramble shipping vectors as the template for PCR. Amplify the sponge region using previously described PCR primers5. Note that forward and reverse PCR primers contain restriction enzyme sequences to permit ligation into α-MHC-pEGFP-C1. Perform PCR as described above (step 2.2.2). Purify the PCR products prior to digestion as described (step 2.2.3) and eluted them with water for digestion. For primer sequences, see Table 1.
    2. Digest the sponge DNA for ligation. A 50 μL digestion reaction contains 1x digest buffer, the gel-purified PCR reaction from step 3.2.1, and 5 μL of both EcoRI and KpnI enzymes. Add all components to a microcentrifuge tube and gently mix them by flicking it. Spin the tube for 10 s in a microcentrifuge to collect the reaction at the bottom. Incubate the reaction in a 37 °C water bath for 15 min.
    3. Purify the digested PCR sponge using a PCR clean-up column as described previously (step 2.2.1).
  3. Ligate the miR-181-sponge into α-MHC-pEGFP-C1 vector
    1. Set up a 21 μL ligation reaction which contains 1x ligation buffer, 3 μL of digested and purified miR-181-sponge PCR, 1 μL of linearized α-MHC-pEGFP-C1 vector, 1x DNA buffer, and 1 μL of DNA ligase. Perform the ligation at room temperature for 5 min and then place it on ice.
    2. Transform 50 μL of XL1-blue competent cells with 2 μL of ligation mix. Use the transformation and plating protocols as described above (step 2.2.5). Perform a colony PCR to identify Kan resistant bacteria which contain the correct α-MHC-pEGFP-miR-181-sponge sequence. Design screening primers to the PCR across the ligation junction. See Table 1 for primer sequences.
    3. Amplify and sequence the vector. Expand the PCR positive clones in LB-Kan broth and in plasmids purified using standard mini-prep plasmid purification protocols. Perform a DNA sequencing to verify the plasmid expressing the desired DNA sequences.

4. Generation of Stable H9c2 miR-181-sponge Expressing Cells

  1. Growth and maintenance of H9c2 cells
    1. Culture the H9c2 cells in Dulbecco's Modified Eagle's Medium (DMEM) containing fetal bovine serum (FBS) to a final concentration of 10%. Sub-culture the cells using standard protocols and grow them at 37 °C in 5% carbon dioxide.
  2. Transfection and selection of H9c2 α-MHC-pEGFP-C1-miR-181-sponge expressing cells
    1. Perform an electroporation of H9c2 cells for the best results. Transfect rat myoblasts (H9c2) with either a scramble sponge (a 284-nt-long scramble sequence which replaces the miR-181-sponge-sequence) or the α-MHC-pEGFP-miR-181-sponge construct with an electroporator.
      1. Briefly, transfect 4x10-5 cells with 2 μg of plasmid DNA (in 5 µL of H2O) and 100 µL of a solution compatible with the electroporation device. Use an electroporation program of DS-120 to transfect the H9c2 cells.
      2. Incubate the transfected cells in the cuvette for 10 min at room temperature. Add 500 µL of DMEM directly to the cuvette. Gently transfer the total cuvette content to a well of a 6-well plate.
    2. Select for GFP positive cells after 48 h post-transfection by FACs. Plate only the GFP-expressed cells into a 6-well plate with an addition of neomycin (G418) (400ng/mL) to the complete growth media.
      Note: Prior to the G418 selection, a kill curve should be established to determine the amount of neomycin required for the selection. For H9c2 cells, 400 ng/mL of G418 resulted in an approximately 80% death rate of non-plasmid transfected H9c2 cells after 24 h and was considered to be the desired working concentration of G418 for these cells. The cell line was considered stable after growth was maintained in G418 for 16 days.
    3. Perform the FACs sorting of the G418 stable cells using the expression of GFP as a marker for a sponge positive selection. Seed 5 - 10 cells in each well of a 96-well plate by flow-sorting. Expand the monoclonal cells from the 96-well plate to 24-well plates over several passages. Use freezer stocks of cells from passage 3 (P3) cells as the starting point for subsequent experiments.
    4. Perform all cell-line-based experiments with low-passage cells between P5 and P10.

5. Synthesis and Purification of Sponge-nanoparticles (miR-181-sponge Nanoparticles)

  1. Prepare the liposomal nanoparticles by dissolving cationic amphiphile (DOTAP) and co-lipids, cholesterol and DSPE-PEG-OMe, in a 5:5:0.1 mM ratio, respectively, in a mixture of chloroform and methanol in a glass vial.
  2. Remove the organic solvent at 44 - 45 °C, under a vacuum by using a rotary evaporator, followed by a gentle flow of moisture-free nitrogen. Keep the remaining dried film of lipid under a high vacuum for 8 h.
  3. Prepare a mixture by adding 5% glucose to the vacuum-dried lipid film. Leave it overnight to allow this mixture to hydrate the film. Vortex the vial for 2 to 3 min at room temperature in order to produce multilamellar vesicles with an occasional shaking in a 45° C water bath.
    1. Sonicate the multilamellar vesicles to prepare small unilamellar vesicles in an ice bath for 3 - 4 min until clarity can be observed, at a 100% duty cycle and 25 W output power.
  4. Mix either a scramble sequence or a miR-181-sponge sequence cloned into the pEGFP(α-MHC) vectors and liposome on a 1:3 charge ratio basis to form the nanovector4.
    Note: An electrostatic complex of positively-charged liposomal nanoparticles and negatively-charged plasmid DNA is known as a nanovector.

6. Systemic Delivery of Sponge-nanoparticles and Validation of In Vivo Effects

  1. Systemic delivery of sponge-nanoparticles in rats
    1. Use Sprague-Dawley (SD) rats. Inject the rats intravenously with the miR-181-sponge nanovector or scramble nanovector through the tail vein. Use SD male rats, which are about 200 g in body weight.
    2. Perform the tail vein injection using a dose of 4 mg of nanovector/kg body weight and repeat this using a regimen of 6 tail vein injections over 2 weeks. Following the repeated nanovector delivery, allow a 1-week period (days 15 - 21) for the expression of the vector in vivo.
    3. Confirm the optimization by imaging the GFP signal before, during, and after the nanovector delivery. Analyze the GFP signal by tomographic imaging software, which generates semi-quantitative data.
  2. Validation of in vivo miR-181 inhibition by the miR-181-sponge
    1. Perform western blot in order to demonstrate the expression of a functional miR-181-sponge in the heart tissue.
      Note: For this study, mt-COX1 expression was examined.
  3. Functional consequences of in vivo miR-181-sponge expression in the heart
    1. Perform a two-dimensional, M-mode, and Doppler echocardiography during and after the nanovector delivery to analyze the cardiac function and morphological changes. For a better scanning capacity in the rodent hearts, use an ultrasound system equipped with a 15 MHz linear array transducer.
    2. Anesthesia may influence the cardiac contractility; therefore, perform the delivery of the miR-181-sponge nanovector or scramble nanovector to animals that are conscious and without any anesthetic reagent during the echocardiography process. Please see Das, et al.4 for further clarification.

Results

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...

Discussion

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...

Disclosures

The authors have nothing to disclose.

Acknowledgements

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's Hospital.

Materials

NameCompanyCatalog NumberComments
pEGFP-C1 vectorAddgene6084-1
In-fusionClontech121416
QIAprep MiniprepQiagen27104
QIAquick Gel Extraction KitQiagen28704
miR-181-sponge synthesisIntrogen GeneArtcustome made
PCR primersIntegrated DNA Technologiescustome
EcoRI enzymesNew Endland BiolabsR0101S
KpnI enzymesNew Endland BiolabsR0142S
Rapid DNA Ligation KitSigma-Aldrich11635379001
H9c2 cellsATCCCRL-1446
DMEM MediaThermo Fisher Scientific11965092
Fetal Bovine SerumThermo Fisher Scientific10082139
Nucleofector 2b DeviceLonzaAAB-1001
Nucleofector Kits for H9c2 (2-1)LonzaVCA-1005
G418, GeneticinThermo Fisher Scientific11811023
FACSAria II Flow cytometerBD Bioscience644832
Branson 450 sonifierMarshall ScientificEDP 100-214-239
The Xenogen IVIS Spectrum optical imaging deviceCaliper Life Sciences
Anti-MTCO1 antibodyAbcamab14705
α-tubulin antibodyAbcamab7291
Sequoia C256 ultrasound systemSiemens

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