Characterization of extracellular circulating microRNA

By A. Turchinovich, Ludmila Weiz, Anne Langheinz, B. Burwinkel. Nucleic Acids Research, 2011

1,887 citations recorded by the source corpus.

MicroRNAs (miRNAs), a class of post-transcriptional gene expression regulators, have recently been detected in human body fluids, including peripheral blood plasma as extracellular nuclease resistant entities. However, the origin and function of extracellular circulating miRNA remain essentially unknown. Here, we confirmed that circulating mature miRNA in contrast to mRNA or snRNA is strikingly stable in blood plasma and cell culture media. Furthermore, we found that most miRNA in plasma and cell culture media completely passed through 0.22 µm filters but remained in the supernatant after ultracentrifugation at 110 000g indicating the non-vesicular origin of the extracellular miRNA. Furthermore, western blot immunoassay revealed that extracellular miRNA ultrafiltrated together with the 96 kDa Ago2 protein, a part of RNA-induced silencing complex. Moreover, miRNAs in both blood plasma and cell culture media co-immunoprecipited with anti-Ago2 antibody in a detergent free environment. This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins. We hypothesize that extracellular miRNAs are in the most part by-products of dead cells that remain in extracellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex. Nevertheless, our data does not reject the possibility that some miRNAs can be associated with exosomes.

Subfields: Cancer Research, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/c7bcd638abaae6019a5afedda1f8ff6dba836908

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Citations in the corpus

Circulating microRNAs in Sera Correlate with Soluble Biomarkers of Immune Activation but Do Not Predict Mortality in ART Treated Individuals with HIV-1 Infection: A Case Control Study (2015, methodology): “However, miRNAs are extremely stable and able to avoid RNase activity through two methods: first, by associating with argonaute (AGO) proteins (>90% of circulating miRNAs appear to be AGO bound) [29, 30]; and second, by being Circulating miRNAs in ART Treated Individuals with HIV-1 Infection”
Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation (2018, background): “While several studies found that the majority of extracellular miRNAs are not associated with exosomes/microvesicles but are instead bound to AGO2 (118, 125), another study reported that extracellular miRNAs are present predominantly in exosomes in human serum and saliva (120).”
Quantitative and stoichiometric analysis of the microRNA content of exosomes (2014, background): “However, we and other groups have found that, in healthy individuals, many cell-free circulating miRNAs are present in soluble form and complexed with proteins, such as Argonaute2 (Ago2) (27, 28).”
An overview of microRNAs (2015, neutral): “lipid exosomes protecting them from degradation[221-227].”
The Majority of MicroRNAs Detectable in Serum and Saliva Is Concentrated in Exosomes (2012, result): “In contrast to two recent papers [10,11] which claimed that the majority of miRNAs found in plasma and serum is present primarily outside exosomes here we demonstrate that miRNAs in serum and saliva exist primarily inside exosomes and that using exosomal fraction increases the sensitivity of miRNA d”
Matrix as an Interstitial Transport System (2014, background): “The impermeability of miRNA-containing microvesicles and exosomes to RNases,(153,154) and high stability of Ago2 proteins(155) have been proposed to protect the miRNA in the extracellular environment.(156,157) Interestingly, some miRNAs (eg, let-7a) are associated exclusively with vesicles, whereas ”
The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling (2014, background): “We analysed the presence of Argonaute-2 (Ago2) protein, a member of the RNA-induced silencing complex and a well-characterized extracellular RNA-binding protein (28,29), in all exosome preparations by Western blot analysis (Fig.”
Standardization of sample collection, isolation and analysis methods in extracellular vesicle research (2013, neutral): “EV and other vehicles, such as protein complexes (13,14)”
Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper (2017, methodology): “9) between intracellular and extracellular miRNA profiles,[46,73] while others found weaker cor-”