Ethics Statement

Animal experiments in this work were limited to the harvest of tissues from humanely euthanized animals. The number of animals used was kept to the absolute minimum necessary to insure data quality (5 animals per group). The Regulus Therapeutics Inc. Institutional Animal Care and Use Committee approved all procedures. Briefly, mice were euthanized by exposure to isoflurane (5% v/v) until one minute after breathing stopped. Euthanasia was confirmed by cervical dislocation.

Transgenic and wild-type Animals

miR-21^-/- animals were generated as described previously [19]. Age and gender-matched C57BL6 wild-type animals used in these studies were purchased from Jackson Laboratories.

Anti-miR administration

Age and gender-matched adult C57BL6 mice were administered anti-miR-21 or anti-miR-122 in Phosphate-Buffered Saline (PBS) solution by subcutaneous injection following the dosing regiment as described for each experiment in the Results section. Anti-let-7, anti-miR-21 and anti-miR-122 compounds are complementary to the 5′-end of let-7, miR-21 or miR-122, respectively, with a full phosphorothioate backbone and sugar modifications such as such as constrained Ethyl (cEt)/DNA or Fluoro/Methoxyethyl at the 2′ position of the sugar.

For cell culture experiments, anti-miRs were transfected using the Lipofectamine RNAiMax reagent (Life Technologies), at the indicated concentration, following the manufacturer's instructions.

Immunopurification of Argonaute complexes and Northern Blot Analysis

Immunopurification of Argonaute from liver extracts and cultured cells was performed using the 4F9 antibody [26] essentially as described previously [3], [27], [28]. Briefly, 100–200 mg of fresh or flash-frozen liver samples were homogenized with a Dounce homogenizer in 2 ml of buffer B [20 mM Tris-HCl pH 8.0, 140 mM KCl, 5 mM EDTA pH 8.0, 0.5% NP-40, 0.1% deoxycholate, 100 U/ml Rnaseout (Life Technologies), 1 mM DTT and 1X Halt protease inhibitor cocktail (Pierce)]. The crude lysate was centrifuged at 1,000 g for 5 mins at 4°C. The supernatant was transferred to a new tube and centrifuged at 16,000 g for 5 mins at 4°C. The S16 supernatant was adjusted to 2 ml with buffer B and incubated with 10–20 ug of 4F9 antibody conjugated to epoxy magnetic beads (M-270 Dynalbeads, Life Technologies) for 2 hours at 4°C with gentle rotation. The beads were then collected by magnets, lysate was removed and the beads were washed three times five mins with 2 ml of buffer C [20 mM Tris-HCl pH 8.0, 140 mM KCl, 5 mM EDTA pH 8.0, 40 U/ml Rnaseout (Life Technologies), 1 mM DTT and 1X Halt protease inhibitor cocktail (Pierce)]. Following immunopurification, RNA and anti-miRs were extracted using standard phenol chloroform extraction methods. To isolate only the anti-miR compounds, RNA was hydrolyzed in the presence of 0.1N NaOH at 65°C for 15 mins, neutralized with 0.1 volume 1 M HEPES buffer. Oligonucleotides were precipitated with 0.1 volume sodium acetate and 5 volumes isopropanol at -20°C for 1 hr, centrifuged at 12,000 g for 15 mins at 4°C, washed with 70% ethanol.

anti-miRs were subjected to Northern blot analysis using Criterion 15% Tris-borate-EDTA (TBE)-Urea precast gels (Bio-Rad) following the manufacturer's recommendations. Oligonucleotides were transferred to N+ Nylon membrane (GE Healthcare), and UV-crosslinked following the manufacturer's protocol. The membrane was incubated with 3X saline-sodium citrate (SSC) solution with 0.1% sodium dodecyl sulphate (SDS). A Starfire (IDT DNA) probe for the sequence complementary to the anti-miR was prepared according to the manufacturer's instructions using [α-32P]-dATP and added to the hybridization solution for an overnight incubation at 42°C. The membrane was then washed three times in 2X SSC with 0.1% SDS, exposed to the phosphor imaging screen K (Bio-Rad) and developed using the Personal Molecular Imager FX Plus (Bio-Rad).

Association of biotinylated compounds with Argonaute complexes and competition binding assays

Liver extracts were prepared as described above and the total protein concentration was adjusted to 10 mg/ml post centrifugation at 16,000 g for 15 mins at 4°C. Streptavidin-conjugated magnetic beads (MyOne T1, Life Technologies) were equilibrated with lysis buffer B and used to preclear the liver extracts for 30 mins at 4°C on a rocking mixer. The pre-cleared lysate was incubated with increasing amounts of 5′-biotinylated compounds for 2 hr at 4°C on a rocking mixer and washed three times for 5 mins with 1 ml of wash buffer. The samples were boiled in 1X LDS Sample buffer (Life Technologies) and loaded onto a pre-cast SDS-PAGE gel (Bio-Rad), transferred onto a PVDF membrane (Bio-Rad) and blocked using the manufacturer's instructions. Argonaute proteins were visualized using the mouse monoclonal anti-EIF2C2 antibody (2E12-1C9, Sigma-Aldrich) and a goat-anti-mouse secondary antibody conjugated to Horseradish Peroxidase (Jackson Immunoresearch labs), developed using the ECL Prime kit (GE Healthcare), digitized by the Fluorochem Q device (Cell Biosciences) and analyzed by the ImageJ software (NIH).

For the competition binding assay, Argonaute complexes were purified from liver or cell extracts using the 4F9 antibody or control IgA and immobilized on a 96-well electrochemiluminescence plate (Meso Scale Discovery), blocked, washed, incubated with an oligonucleotide complementary to the miRNA sequence (IDT DNA) and conjugated with a Sulfo-TAG (MSD) or a chemically modified oligonucleotide, following the manufacturer's instructions. Luminescence was quantified using the Sector Imager 2400 device (Meso Scale Discovery). Background levels were determined based on the luminescence levels of control IgA immunopurifications and were subtracted from the luminescence signal.

RNA extraction and quantification of mRNAs

RNA from liver or cell line samples was extracted using the Qiagen RNeasy Kit or Qiagen miRNeasy Kit, following the manufacturer's instructions. mRNA levels were analyzed by qRT-PCR using Taqman One Step RT-PCR kit on a Viia 7 thermocycler (Life Technologies), relative to the GUSB housekeeping gene and processed using the ΔΔC_t method.

Isolation and Quantification of miRNAs from anti-miR treated livers

Total RNA was isolated from 100 ul of S16 lysate (total RNA, post-16,000 g centrifugation) or 100 ul of resuspended Argonaute immunopurified samples by double extraction with Phenol:Chloroform:Isoamyl Alcohol (25∶24∶1, v/v) and one extraction with chloroform. 5 volumes of isopropanol, 1/10^th volume 3 M sodium acetate and 1 ul glycoblue (Life Technologies) were added to the extracted samples and RNA was precipitated by a 30 min centrifugation at 12,000 g at 4°C. The pellet was washed with 200 ul of ice-cold 70% ethanol, dried, and resuspended in 50 ul of RNAse-free water.

For the glyoxal treatment [29], 6 M glyoxal (Sigma) was deionized with 1 w/v AG 501-X8 resin (Bio-Rad) for 1 hour at RT on a rotator. Glyoxal working solutions were prepared with 5 ml deionized glyoxal, 6 ml DMSO and 10 mM sodium phosphate, pH 6.5. Aliquots were stored at −80°C. Equal volume of glyoxal working solution was added to RNA solutions and samples were incubated at 50°C for 30 min. Glyoxal was removed via miRNeasy kit (Qiagen) following manufacturor's protocol. Equal volume of reverse glyoxal solution (100 mM Tris-HCl, pH 8.0, 1 M KCl, 1 mM EDTA pH = 8.0) was added to purified RNA and samples were incubated overnight at 60°C. RNA was then purified by miRNeasy kit.

Nanostring hybridization was performed with nCounter mouse V1.2 miRNA expression assay (Nanostring) using 100 ng of total RNA or the equivalent miRNA content of 100 ng total RNA in Argonaute-immunopurified RNA, as measured by qRT-PCR of let-7A and miR-21.

Melting curves

The melting temperatures for anti-miR-21-A and anti-miR-21-B oligonucleotide were determined as previously described [30]. The temperatures listed are an average of three experiments.

Microarray Analysis

mRNA was profiled using Mouse Genome 430 2.0 array Human Genome 133 Plus v. 3 (Affymetrix) per manufacturer's instructions in triplicate. Cumulative distribution fractions were calculated as described previously [31].

Anti-miRs associate with the Argonaute complex in vivo

To gain insight into the mechanism of action of miRNA inhibitors, we sought to determine whether the anti-miRs physically associate with the Argonaute complex in vivo. To achieve this we performed Argonaute immunopurifications in liver lysates from anti-miR-122-treated mice and determined the levels of anti-miR associated with Argonaute (Figure 1a). Purified total RNA (and anti-miR) from input and Argonaute immunopurification samples were subjected to conditions that hydrolyze RNA without compromising the chemically modified anti-miRs, which were detected by Northern blot analysis. Anti-miRs can be detected in the input liver lysate, as expected, but also in the Argonaute immunopurifications, establishing that anti-miRs physically associate with the Argonaute complexes in vivo. Under typical immunoprecipitation conditions, we isolate approximately 30–40% of the Argonaute protein, and similar amounts of mature miRNAs (Figure S1) suggesting that the vast majority of mature miRNAs are associated with Argonaute and can be targeted by our anti-miRs.

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Figure 1. Anti-miRs specifically associate with the Argonaute in vivo and in vitro, only in the context of the cognate target miRNA.

a) Northern analysis of anti-miR-122 in input lysate and in immunopurified Argonaute-containing complexes (IP) from liver tissue of animals dosed subcutaneously, three days prior, with 1, 3 or 10 mg/kg of the compound. Representative experiment shown from 2 independent experiments. b) Western analysis of the levels of Argonaute associated with biotinylated compounds in liver lysates from wild-type and miR-21-deficient animals. 10,3, or 1 pmoles of the 5′-biotinylated compounds were incubated with liver lysates, purified with streptavidin beads and the associated Argonaute proteins were visualized with an anti-Argonaute antibody. Representative experiment shown from three independent experiments. c) Quantification of Argonaute levels associated with the compounds from panel (b). d) Luminescence levels of a miR-21 probe bound by immunopurified miRNA:Argonaute complexes from wild-type or miR-21-lacking liver extracts (miR-21^-/-), in the presence of increasing amounts of anti-miR-21. A miR-122 probe in the presence of increasing amounts of anti-miR-122 was used as a positive control for the assay. Representative experiments shown from 3 independent experiments (n = 3).

https://doi.org/10.1371/journal.pone.0100951.g001

Anti-miRs associate specifically with the target miRNA in Argonaute

MiRNA inhibitors are designed to hybridize with the target miRNA sequence with high affinity [12], [13], [23]. To verify that the physical association of the anti-miR to the Argonaute complex is achieved only in the context of the target miRNA, we performed Argonaute/anti-miR binding assays in liver lysates from wild-type and miR-21-deficient mice [19] (Figure 1b). Increasing levels of the 5′-biotinylated anti-miR-21 were incubated with liver lysates from wild-type and miR-21 knockout mice (miR-21^-/-) and purified with streptavidin beads. The levels of Argonaute protein associated with the purified biotinylated oligo were visualized with Western blot analysis (Figure 1b,c). Argonaute proteins co-purify with biotinylated anti-miR-21 oligos in wild-type lysates but not in liver lysates lacking miR-21, suggesting that anti-miR-21 association with Argonaute is specifically mediated through the target miR-21. As a positive control, the biotinylated anti-miR-122 oligo associated with Argonaute in both wild-type and miR-21-deficient lysates. Interestingly, the Argonaute/anti-miR-21 association is saturated at 3 pmoles, while the Argonaute/anti-miR-122 association is not saturated (Figure 1b,c), reflecting the higher levels of miR-122 expressed in the liver lysate compared to miR-21.

We also verified our findings by employing a distinct, electrochemiluminescence-based competition binding assay (Figure 1d and S2). Briefly, Argonaute complexes were immunopurified from wild-type and miR-21-deficient liver lysate and incubated with a Ruthenium complex-labeled oligo complementary to the miR-21 sequence, in the presence of increasing amounts of the anti-miR-21 compound. Association of the compound with the miRNA:Argonaute complex in the wild-type lysate led to decrease in the Argonaute-associated luminescent probe as both probe and increasing amounts of compound compete for Argonaute association. In extracts lacking the target miRNA (miR-21^-/- extract), the probe fails to associate with Argonaute, leading to background levels luminescence. Performing the experiment with a miR-122 probe and anti-miR-122 compounds yielded equivalent results for both lysate sources and served as a positive control for the assay. Taken together, our data suggest that anti-miRs associate with the miRNA:Argonaute complex only in the context of the targeted miRNA.

Anti-miRs associate with the miRNA:Argonaute complexes via the seed region

The seed region of the miRNA, nucleotides 2-8, is a major determinant of miRNA targeting [1]. To investigate the importance of seed complementarity in the ability of anti-miRs to associate with the miRNA:Argonaute complex, we performed binding assays with 5′-biotinylated compounds with perfect and mismatched seed sequences (Figure 2). Not surprisingly, mismatches in the seed region abolished the binding ability of the anti-miR-122 for the Argonaute:miR-122 complex (Figure 2a) as evidenced by the inability of purified biotinylated mismatch compounds to co-purify with Argonaute. On the contrary, seed-matched compounds associated strongly with Argonaute complexes as visualized by Western blot analysis. We confirmed these results independently with the electrochemiluminescence-based competition-binding assay we developed (Figure 2b). As expected, the increasing concentrations of mismatched compound fail to compete with a miR-122-complementary probe, suggesting that the mismatch compound fails to associate with the miRNA:Argonaute complexes. However, the seed-matched version of the compound competed with the luminescent probe for association with Argonaute, as demonstrated by the reduced levels of Argonaute-bound luminescence with increasing concentrations of the compounds.

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Figure 2. The seed region is an important determinant for anti-miRs to associate with the miRNA:Argonaute complexes in vitro and inhibit the target miRNA in vivo.

a) Western analysis of the levels of Argonaute from liver lysates that associate with an anti-miR-122 compound with perfect seed matching (match) or with 3 mismatches in the seed region (mismatch). 10,3, or 1 pmoles of the 5′-biotinylated compounds were incubated with liver lysates, purified with streptavidin beads and the associated Argonaute proteins were visualized with an anti-Argonaute antibody. Representative experiment of 3 independent experiments. b) Luminescence levels of a miR-122 probe bound by Argonaute in the presence of an anti-miR-122 compound with perfect seed matching and its seed-mismatched counterpart (3 mismatches) or an unrelated negative control sequence. Match and mismatch sequences are as in (a). Representative experiment shown from 3 independent experiments (n = 3). c) mRNA levels of the miR-122-regulated Aldoa transcript in livers of mice treated with PBS or anti-miR-122 compounds with mismatches as shown in the table. Mice were treated subcutaneously for ten days with 25 mg/kg of the indicated compound. Results are shown as percentage of change over the PBS-treated animals ± s.d. Representative experiment from 2 independent experiments (n = 5). d) Aldoa mRNA levels in animals treated with anti-miR-122 compounds targeting the seed plus 6 complementary or random nucleotides. PBS controls or anti-miR solutions were administered subcutaneously at 25 mg/kg. Representative experiment from 2 independent experiments (n = 5).

https://doi.org/10.1371/journal.pone.0100951.g002

To further establish the importance of targeting the seed region of the miRNA in order to inhibit its function in vivo, we generated a series of anti-miR-122 with the same chemical modifications but with three mismatches in various locations along the anti-miR sequence (Figure 2c). The effect of anti-miRs on the derepression of Aldoa, a direct target of miR-122 [21], [32], was used to evaluate the ability of the compounds to inhibit miR-122 function. Mismatches in the compound sequence that corresponds to the target miRNA seed region, nucleotides 1–3, 2–4, 5–7 completely abolished the ability of the anti-miR-122 compound to inhibit miR-122 and derepress Aldoa, exhibiting approximately the same effect as injections of PBS solution. Mismatches in nucleotides 8–10 slightly diminished the potency of the anti-miR-122 compound, most likely due to the mismatched nucleotide 8, the last nucleotide in the seed region. However, mismatches in the non-seed region are well tolerated, yielding results equivalent to fully complementary miRNA inhibitors, typically a 4-fold derepression of the Aldoa target.

Since binding of the anti-miR to the target miRNA:Argonaute complex appears to be mediated by the miRNA seed, we investigated whether perfect complementarity with the target miRNA is required for inhibiting miRNA function. We generated two 14 nt-long anti-miR-122 compounds, encompassing the seed sequence plus 6 nucleotides, fully complementary or with a random sequence (Figure 2d), in order to control for pharmacokinetics properties in vivo. The ability of both compounds to derepress the miR-122 target Aldoa is similar (Figure 2d), suggesting that perfect complementarity outside the seed is not required for target engagement in vivo. Collectively, these results indicate that anti-miRs associate with the target miRNA:Argonaute complexes via base-pairing to the seed region of the target miRNA and there is no requirement for complementarity outside the seed sequence.

The ability of anti-miRs to target miRNA:Argonaute complexes and inhibit their function in vitro and in vivo is affected by chemical modification patterns

A number of distinct chemical modifications can be incorporated into the design of an anti-miR to convey desired properties, such as improved stability, potency and pharmacokinetic properties [12], [15], [32]. To determine if the chemical modification pattern in anti-miRs can affect their ability to bind to their target miRNA in the Argonaute complex and thus influence their capacity to inhibit the miRNA:Argonaute complex in vitro and in vivo, we generated two anti-miR-21 compounds, of the same sequence but with slightly different chemical modification placement in 2 positions, termed compounds A and B, as shown in Figure 3a. Anti-miR-21-A and B contain a mix of constrained ethyl-modified (cEt) and DNA bases but differ in their placement at two specific locations as shown in the figure. For pairing to an adenosine in the target miRNA, we employ either a constrained ethyl uracil or a DNA thymidine. Both compounds have similar binding affinity to miR-21, as determined by the melting curve analysis with Tm = 72.7 and Tm = 72.2, respectively (Figure S3). We evaluated the ability of the compounds to associate with Argonaute in vitro (Figure 3a,b) and to derepress miR-21 targets in cell culture and in vivo (Figure 3c,d). Anti-miR-21-A barely co-purifies with Argonaute complexes from liver lysates, even at high levels of spiked 5′-biotinylated compound, while compound B can strongly associate with Argonaute at all levels (Figure 3a). According to the more sensitive electrochemiluminescence-based competition assay (Figure 3b), anti-miR-B is more potent at associating with the Argonaute-bound miR-21 than anti-miR A, as evidenced by the dramatic decrease in luminesce (anti-miR-A logIC₅₀ = −0.42 pmoles, anti-miR-B logIC₅₀ = −1.96 pmoles). The unrelated target sequence (let-7), was used as a negative control and did not compete with the probe. On the other hand increasing amounts of unlabeled probe competed with the labeled probe as expected, serving as a positive control.

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Figure 3. Subtle changes in the chemical modification pattern can affect anti-miR association with Argonaute and anti-miR activity in vitro and in vivo.

a) Western analysis of the levels of Argonaute associated with anti-miR-21-A or anti-miR-21-B compounds, in liver lysates. These two compounds share the same sequence and chemistry, a phosphorothioate backbone with DNA and constrained Ethyl (red) bases. 10,3, or 1 pmoles of the 5′-biotinylated compounds were incubated with liver lysates, purified with streptavidin beads and the associated Argonaute proteins were visualized with an anti-Argonaute antibody. Samples from the same gel. Representative experiment from 3 independent experiments. b) Luminescence levels of a miR-21 probe bound by Argonaute in a competition binding assay with increasing amounts of anti-miR-21-A, anti-miR-21-B, unlabeled probe or an unrelated sequence as control. miRNA:Argonaute complexes were purified from HeLa cells. Representative experiment from 3 independent experiments (n = 3). c) mRNA levels of the miR-21-regulated ANKRD46 gene in U87 cells transfected with increasing concentrations of anti-miR-21-A or anti-miR-21-B compounds. Results are shown as fold change over mock transfected ± s.e.m. Representative experiment from 2 independent experiments (n = 5). d) mRNA levels of the miR-21-regulated Rnf167 transcript in livers of mice treated with PBS or the anti-miR-21 compounds A or B. Mice were injected subcutaneously once with 50 or 100 mg/kg of anti-miR-21-A or B, RNA was extracted from liver samples three days post-injection, and analyzed for Rnf167 mRNA levels by qPCR. Results are shown as fold change over the PBS-treated group of animals ± s.d. Representative experiment from 2 independent experiments (n = 5).

https://doi.org/10.1371/journal.pone.0100951.g003

Having established that subtle changes to the modification pattern can affect the ability of the anti-miR to target the Argonaute-bound miRNA, we sought to determine their effect on derepressing miR-21 mRNA targets in cell culture and in vivo. For this, we transfected U87 cells with increasing concentrations of anti-miR-21-A or anti-miR-21-B and examined their effect on the derepression of ANKRD46 (Figure 3c), a target of miR-21 [33]. Treatment with anti-miR-B derepressed ANKRD46 in a dose-response manner, increasing the mRNA levels ∼3.5 fold over mock-transfected. At the same concentration (17 nM), anti-miR-21-A barely increased ANKRD46 mRNA levels. We also determined the effect of these compounds on the derepression of Rnf167, a miR-21 target [34], in vivo (Figure 3d). Anti-miR-21-B treatment led to a dose-response derepression of Rnf167 of ∼1.6-fold at the high dose, while anti-miR-21-A response was at control levels. Taken together, these data indicate that alterations in the modification pattern of anti-miRs can affect their ability to target miRs in the Argonaute context and to inhibit the target miRNA:Argonaute function in vitro and in vivo.

Anti-miR treatment leads to decreased association of Argonaute with target mRNAs increasing their abundance

Having established that anti-miRs associate with the target miRNA in the context of Argonaute we hypothesized that the anti-miR association with the miRNA:Argonaute complex prevents Argonaute-bound miRNAs from binding and regulating target mRNAs. We tested this hypothesis in vivo and in vitro, by performing Argonaute immunopurifications in liver lysates from mice treated with anti-miRs or from cell lines transfected with anti-miRs (Figure 4 and S4). We profiled total RNA and Argonaute-immunopurified fractions via microarray hybridization and focused on changes in mRNAs with with 6–7- or 8-nt seed matches as described previously [3], [31] (Figure 4a,b). mRNA transcripts with seed matches in the 3′UTR were derepressed in total mRNA compared to PBS-treated controls. Transcripts with 8-mer seed matches in the 3′UTR exhibited the highest derepression, followed by 7- and 6-mer matches, while seed-matches in the coding sequence (CDS) had the smallest effect (Figure 4a). In contrast, transcripts with 8-mer seed matches associated less with Argonaute in the presence of the anti-miR (Figure 4b). The levels of established miR-122 target genes Aldoa and Cd320 [21], [35], [36] in the total lysate and in the Argonaute-associated fraction were also quantified in these samples by qPCR (Figure 4c,d). Treatment with the anti-miR-122 reduced the fraction of the targets associated with Argonaute compared to the PBS-treated samples, ∼6- and ∼4-fold respectively in a dose-dependent manner (Figure 4c,d). qPCR analysis of the relative abundance of Aldoa and Cd320 mRNA transcripts compared to PBS-treated controls revealed a ∼4-fold increase for both transcripts.

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Figure 4. Anti-miR treatment decreases the levels of Argonaute-bound mRNA targets, increasing their stability and abundance in vivo.

a and b) Cumulative-distribution fraction plots (CDF) depicting the mRNA fold change in total RNA (a) and in the Argonaute immunopurified (Ago) fraction (b) from liver lysates of animals dosed with with 10 1 mg/kg of anti-miR-122 as compared to PBS-treated controls as determined by microarray analysis. c and d) Total and Argonaute IP (Ago) RNA was assayed for the miR-122 targets (c) Aldoa and (d) Cd320. Mice were treated subcutaneously with 1, 3 or 10 mg/kg of an anti-miR-122 compound or PBS and livers were collected three days later. Results are displayed as fold-change±s.e.m.. Representative experiment from 2 independent experiments shown (n = 3).

https://doi.org/10.1371/journal.pone.0100951.g004

We obtained similar results when we performed Argonaute immunopurification in HeLa cell lines transfected with anti-let-7 (Figure S4). The presence of the anti-miR led to a dramatic increase in the levels of the mRNA transcripts with 8-, 7- or 6-mer seed matches, while their association with Argonaute was dramatically reduced (Figure S4a,b). For the established let-7 targets IGF2BP1 and HMGA2 [37], [38] transcript abundance increased while Argonaute association was inhibited in a dose-response manner by the anti-let-7 treatment as determined by qPCR (Figure S4c). These results are consistent with the hypothesis stated above, that in the presence of an anti-miR, mRNA transcripts are no longer targeted and bound by the miRNA:Argonaute complexes which leads to an increase in their stability and thus their abundance; on the other hand, the miRNA:Argonaute complex is now occupied with the high-affinity anti-miR.

Anti-miRs do not affect the levels of mature miRNAs or their association with Argonaute

To investigate the effect of miRNA inhibitor treatment on the target miRNA and its association with Argonaute complex, we sought to determine if anti-miR treatment containing a mixture of DNA and constrained ethyl chemistry leads to degradation and/or ejection of the target miRNA from the Argonaute complex in vivo by performing Argonaute immunopurifications and RNA analysis (Figure 5). By design, anti-miRs form high-affinity duplexes with target miRNAs, a phenomenon that can interfere with miRNA detection obscuring the effect of anti-miR on the miRNA levels [23]. Although competitor-based methodologies have been developed to visualize the target miRNAs in the presence of ant-miRs [23], they are not compatible with quantitative and high-throughput miRNA level analysis. To address this issue, we subjected the immunopurified Argonaute-associated and total RNA fractions to glyoxal treatment [29], that allows the separation of anti-miR and target miRNA. Glyoxal is a dialdehyde that forms a reversible covalent adduct with guanosine, decreasing the affinity for the miRNA and enabling the separation of miRNAs and anti-miRs. This treatment allows for the selective purification of miRNAs using standard silica-membrane–based purification columns. Following this approach, we quantified the levels of miR-122 and let-7a miRNAs in the treated samples using qPCR before (Figure 5a) and after glyoxal treatment (Figure 5b) relative to miR-194 levels. Glyoxal treatment allowed for the detection of miR-122 in the treated samples at the same relative levels as let-7a, which was not targeted by an anti-miR. After establishing that we can accurately and quantitatively detect the levels of targeted miRNAs, we analyzed the global miRNA profile of the anti-miR-122-treated or PBS-treated samples, both for total RNA (Figure 5c and S5a) and immunopurified Argonaute-associated fractions (Figure 5d and S5b). The comparison between miR-122-treated vs. PBS-treated samples for both total RNA and Argonaute-associated fractions indicates that the relative levels of miR-122 remain mostly stable for both total lysate as well as in the Argonaute complex (Figure 5c,d and S5a,b). Taken together, these results suggest that treatment with a constrained ethyl/DNA anti-miR-122 does not affect the stability of mature miRNA levels or the miRNA:Argonaute association, in accordance with previously published results for anti-miRs with 2′-Fluoro and 2′-O-Methoxyethyl groups [23].

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Figure 5. Anti-miRs do not affect the levels of total mature or Argonaute-bound miRNA in vivo.

Total liver RNA isolated from animals 7 days post-subcutaneous administration of anti-miR-122 (10 mg/kg). miRNA levels in total RNA sample, profiled on the Nanostring platform, before (a) and after (b) the glyoxal treatment to separate the miR/anti-miR-122 duplex which interferes with miR-122 detection. c and d) Global miRNA levels, post-glyoxal treatment and profiled on the Nanostring platform, in anti-miR-122-treated animals compared to PBS-treated controls from total RNA (c) or immunopurified Argonaute fractions (Ago) (d). Representative experiments shown from 2 independent experiments (n = 3).

https://doi.org/10.1371/journal.pone.0100951.g005