Extracellular microRNAs exhibit sequence-dependent stability and cellular release kinetics

Multiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4oC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37oC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from similar to 1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties

Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed, with dystrophin uniformly distributed at the sarcolemma of corrected myofibers at all doses. Serum microRNA biomarkers (i.e., miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase levels were restored toward wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e., Young’s modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression and that both serum microRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in DMD

Investigation of extracellular microRNAs and Serum Protein Biomarkers in dystrophic Muscle Disease

Extracellular microRNAs (ex-miRNAs) and serum proteins are promising biomarkers for a variety of pathological conditions, including muscular dystrophies and, Duchenne Muscular Dystrophy (DMD) in particular. miR-1, miR-133 and miR-206 are muscle-specific miRNAs (myomiRs) that regulate myoblast proliferation and differentiation. These myomiRs are highly abundant in serum of DMD patients and dystrophic mdx mice and have therefore been investigated as biomarkers of dystrophic disease. The biological significance of miRNAs present in the extracellular space is currently not well understood. The work presented here demonstrates that ex-myomiRs are selectively released during periods of myogenic differentiation in cell culture and in vivo. Consequently, their release can be independent of dystrophic pathology, thus indicating that the presence of myomiRs in the serum may serve a physiological function in some contexts. In summary, serum myomiR abundance appears to be a function of the regenerative/degenerative status of the muscle, overall muscle mass, tissue expression levels and myomiR stability in serum. These findings have implications both for miRNA biology in normal muscle physiology as well as for the use of ex-myomiRs as biomarkers for DMD. Secondly, size-exclusion chromatography was identified as a useful methodology to fractionate biofluids and thereby separate extracellular vesicles (EV) from protein complexes and lipoproteins. Using this tool, it was demonstrated that ex-myomiR carriers in cell culture supernatant and murine serum are predominantly non-vesicular, and furthermore, that their release is independent of ceramide-mediated vesicle secretion. In addition, it was found that EVs isolated from dystrophic mice are smaller and more numerous compared to EVs derived from wild-type mice, thereby indicating a role for dystrophic pathology in the regulation of vesicle biogenesis and/or secretion. There is currently an urgent need for minimally invasive, therapy-monitoring DMD biomarkers for use in clinical trials. Serum protein biomarkers are desirable since they can typically be quantified by clinical biochemistry assays, which are technically facile and allow for rapid screening of large numbers of patients. Previously, identification of such biomarkers by mass spectrometry has been limited due to analytical challenges resulting from the massive complexity of the serum proteome. Here, an aptamer-based proteomics approach was utilised to profile 1,129 proteins in the serum of wild-type, mdx mice and, mdx mice treated with exon skipping therapy to restore dystrophin expression. A number of novel, therapy-responsive biomarkers were identified, and the leading candidate (ADAMTS5) was also found to be significantly elevated in DMD patient serum. In conclusion, this work demonstrates that myomiR release accompanies myogenic differentiation and shows that serum myomiR levels are influenced by a number of physiological and pathological factors. Furthermore, multiple novel, therapy-responsive protein biomarkers were identified in the serum of the mdx mouse with potential utility in clinical trials for DMD.</p

Investigation of extracellular microRNAs and Serum Protein Biomarkers in dystrophic Muscle Disease

Extracellular microRNAs (ex-miRNAs) and serum proteins are promising biomarkers for a variety of pathological conditions, including muscular dystrophies and, Duchenne Muscular Dystrophy (DMD) in particular. miR-1, miR-133 and miR-206 are muscle-specific miRNAs (myomiRs) that regulate myoblast proliferation and differentiation. These myomiRs are highly abundant in serum of DMD patients and dystrophic mdx mice and have therefore been investigated as biomarkers of dystrophic disease. The biological significance of miRNAs present in the extracellular space is currently not well understood. The work presented here demonstrates that ex-myomiRs are selectively released during periods of myogenic differentiation in cell culture and in vivo. Consequently, their release can be independent of dystrophic pathology, thus indicating that the presence of myomiRs in the serum may serve a physiological function in some contexts. In summary, serum myomiR abundance appears to be a function of the regenerative/degenerative status of the muscle, overall muscle mass, tissue expression levels and myomiR stability in serum. These findings have implications both for miRNA biology in normal muscle physiology as well as for the use of ex-myomiRs as biomarkers for DMD. Secondly, size-exclusion chromatography was identified as a useful methodology to fractionate biofluids and thereby separate extracellular vesicles (EV) from protein complexes and lipoproteins. Using this tool, it was demonstrated that ex-myomiR carriers in cell culture supernatant and murine serum are predominantly non-vesicular, and furthermore, that their release is independent of ceramide-mediated vesicle secretion. In addition, it was found that EVs isolated from dystrophic mice are smaller and more numerous compared to EVs derived from wild-type mice, thereby indicating a role for dystrophic pathology in the regulation of vesicle biogenesis and/or secretion. There is currently an urgent need for minimally invasive, therapy-monitoring DMD biomarkers for use in clinical trials. Serum protein biomarkers are desirable since they can typically be quantified by clinical biochemistry assays, which are technically facile and allow for rapid screening of large numbers of patients. Previously, identification of such biomarkers by mass spectrometry has been limited due to analytical challenges resulting from the massive complexity of the serum proteome. Here, an aptamer-based proteomics approach was utilised to profile 1,129 proteins in the serum of wild-type, mdx mice and, mdx mice treated with exon skipping therapy to restore dystrophin expression. A number of novel, therapy-responsive biomarkers were identified, and the leading candidate (ADAMTS5) was also found to be significantly elevated in DMD patient serum. In conclusion, this work demonstrates that myomiR release accompanies myogenic differentiation and shows that serum myomiR levels are influenced by a number of physiological and pathological factors. Furthermore, multiple novel, therapy-responsive protein biomarkers were identified in the serum of the mdx mouse with potential utility in clinical trials for DMD.</p

Assessment of RT-qPCR Normalization Strategies for Accurate Quantification of Extracellular microRNAs in Murine Serum

<div><p>Extracellular microRNAs (miRNAs) are under investigation as minimally-invasive biomarkers for a wide range of disease conditions. We have recently shown in a mouse model of the progressive muscle-wasting condition Duchenne muscular dystrophy (DMD) that a set of highly elevated serum miRNAs reflects the regenerative status of muscle. These miRNAs are promising biomarkers for monitoring DMD disease progression and the response to experimental therapies. The gold standard miRNA detection methodology is Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR), which typically exhibits high sensitivity and wide dynamic range. Accurate determination of miRNA levels is affected by RT-qPCR normalization method and therefore selection of the optimal strategy is of critical importance. Serum miRNA abundance was measured by RT-qPCR array in 14 week old mice, and by individual RT-qPCR assays in a time course experiment spanning 48 weeks. Here we utilize these two datasets to assess the validity of three miRNA normalization strategies (a) normalization to the average of all Cq values from array experiments, (b) normalization to a stably expressed endogenous reference miRNA, and (c) normalization to an external spike-in synthetic oligonucleotide. Normalization approaches based on endogenous control miRNAs result in an under-estimation of miRNA levels by a factor of ∼2. An increase in total RNA and total miRNA was observed in dystrophic serum which may account for this systematic bias. We conclude that the optimal strategy for this model system is to normalize to a synthetic spike-in control oligonucleotide.</p></div

TRIM protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity

Members of the tripartite interaction motif (TRIM) family of E3 ligases are emerging as critical regulators of innate immunity. To identify new regulators, we carried out a screen of 43 human TRIM proteins for the ability to activate NF-kappaB, AP-1, and interferon, hallmarks of many innate immune signaling pathways. We identified 16 TRIM proteins that induced NF-kappaB and/or AP-1. We found that one of these, TRIM62, functions in the TRIF branch of the TLR4 signaling pathway. Knockdown of TRIM62 in primary macrophages led to a defect in TRIF-mediated late NF-kappaB, AP-1, and interferon production after lipopolysaccharide challenge. We also discovered a role for TRIM15 in the RIG-I-mediated interferon pathway upstream of MAVS. Knockdown of TRIM15 limited virus/RIG-I ligand-induced interferon production and enhanced vesicular stomatitis virus replication. In addition, most TRIM proteins previously identified to inhibit murine leukemia virus (MLV) demonstrated an ability to induce NF-kappaB/AP-1. Interfering with the NF-kappaB and AP-1 signaling induced by the antiretroviral TRIM1 and TRIM62 proteins rescued MLV release. In contrast, human immunodeficiency virus type 1 (HIV-1) gene expression was increased by TRIM proteins that induce NF-kappaB. HIV-1 resistance to inflammatory TRIM proteins mapped to the NF-kappaB sites in the HIV-1 long terminal repeat (LTR) U3 and could be transferred to MLV. Thus, our work identifies new TRIM proteins involved in innate immune signaling and reinforces the striking ability of HIV-1 to exploit innate immune signaling for the purpose of viral replication

Serum time course of putative reference miRNA abundance.

<p>Male C57Bl/10, <i>mdx</i> and Pip6e-PMO treated <i>mdx</i> mice were sacrificed at various ages and serum miRNA levels determined by small RNA TaqMan RT-qPCR. (A) miR-1, (B) miR-133a and (C) miR-206 abundance was normalized to an external spike-in control. All miRNA expression data were normalized to the mean of the 8 week old C57Bl/10 group. Values are mean +/− SEM, n = 3–8.</p

Correlation of Cq values between endogenous miRNAs and the external spike-in control for time course samples.

<p>Plot of raw Cq values from the time course study for cel-miR-39 against (A) miR-16, (B) miR-31, and (C) miR-223.</p