Circulating miRNAs

MicroRNAs are a class of small non-coding RNA molecules 18-25 nucleotides in length that are actively involved in the regulation of gene expression. The action of miRNA is very diverse and is closely associated with many processes occurring in the body. Including with the maintenance of genome stability, immune reactions, differentiation, proliferation, cell apoptosis, both in normal conditions and in various pathologies. And the latter circumstance makes them attractive for researchers and physicians in two directions at once: as a therapeutic target and a potential biomarker for the diagnosis of almost all age-related (and not only) diseases.

The first miRNA, called lin-4, was discovered a quarter of a century ago by scientists from the Harvard University at the nematode S. elegance [1]. Scientists have discovered that the lin-4 gene encoded not two proteins, but two small RNAs - a precursor 61 nucleotides in length and microRNA itself, 22 nucleotides, which suppressed the expression of the nematode gene lin-14, preventing it from developing normally. For a long time, it was believed that miRNA is such an evolutionary exotics, a property of the nematode genome, until seven years later, in 2000, the second miRNA molecule, let-7, was discovered [2]. It suppressed the expression of several genes at once and then was described in a number of living organisms, including humans. And after that, the “dam broke” - the discoveries of miRNA began to follow one after the other.

Today it is known that each miRNA can control many (up to several hundred) genes, and one specific gene can be a target for several miRNAs. How does this small molecule work? miRNAs will silence the gene in several ways. First, they suppress gene expression by interacting with messenger RNA (mRNA). miRNAs are attached to mRNA, which leads to blocking of the translation process (i.e. protein synthesis) and mRNA degradation. The second variant of gene deactivation is transcriptional, when miRNAs within a polyprotein complex cause epiginetic modifications of the genome — DNA methylation and deacetylation and histone methylation.

In addition, another variant of the suppression of miRNA protein synthesis was described by their interaction with repressor proteins that block translation [3]. But at the same time it was found that in some cases, namely, when the cell cycle is stopped, miRNAs can not repress, but activate the translation process. This phenomenon was described in 2007 in the journal Science [4]. But this phenomenon is so rare and not characteristic that in most scientific articles they don’t even mention it.

About 10 years ago, it was first described that miRNAs secreted by one cell type can be transferred to other cell types. This was the reason to assume that in addition to cellular miRNA, extracellular is also present in the body, the so-called. circulating miRNA (circulating miRNA, c-miPHK), which was then detected in blood plasma and other biological fluids. As assumed today, the appearance of c-miPHK in the blood can be the result of both their secretion by cells and the death of the cells themselves during apoptosis and necrosis.

It quickly became clear that miRNAs are resistant to endogenous ribonucleases (RNA killers) and are highly stable in serum and plasma. And their number can be measured with high sensitivity and specificity in several ways, the most common of which is real-time PCR and hybridization with fluorescent probes. And this made it possible to efficiently analyze the levels of circulating miRNA, isolating from biological fluids, and use them as a biomarker in various pathologies.

To diagnose what pathologies can miRNA analysis be used? First of all, these are of course the main age-related diseases - cardiovascular, neurodegenerative and oncological. Thus, numerous studies conducted have shown a close relationship between miRNA levels and cardiovascular diseases (CCPs) - miRNAs play an important role in the development and pathogenesis of CVD. What makes it possible to use them as a diagnostic marker at the earliest stages. Today, several miRNAs are already known that are suitable for this purpose.

Back in 2009, one of the first studies showed that miRNA-208 is specifically produced in the heart cells and its plasma concentration is an accurate indicator of myocardial damage [5]. Later, scientists were able to identify two more miRNAs, miR-423-5p and miRNA-499, which showed good efficacy in the diagnosis of heart failure and acute myocardial infarction [6,7].

During a 10-year period, during a large study in the Nord-Trøndelag Health Study, a number of circulating miRNAs were detected by Norwegian scientists (miR-106a-5p, miR-424-5p, let-7g-5p, miR-144-3p and miR-660-5p,), whose levels can predict future acute myocardial infarction in still healthy people [8]. Moreover, the same work showed that men and women have their own specific miRNAs (miR-424-5p and miR-26a-5p, respectively) associated with the risk of myocardial infarction.

A detailed systematic review of currently known miRNAs, potential biomarkers of cardiovascular diseases, was made in 2018 by Russian cardiologists [9].

It also turned out that in addition to myocardial infarction and heart failure, the analysis of circulating miRNAs in the blood can help in the early diagnosis of stroke, as well as predict the prognostic result in patients. What is especially important for the well-known severe consequences of hemorrhagic stroke, for which, according to doctors, besides microRNA, today there are no established biomarkers for current blood tests in stroke diagnosis [10]

In 2018, a large systematic review was published, which included eight studies , including 572 patients and 431 healthy participants in the control group. According to this review, at least 22 microRNAs are known today, the differential expression of which was registered in the earliest period after acute ischemic stroke [11].

But perhaps the most effective miRNAs have proven to be biomarkers in the early diagnosis of cancer. For example, in 2014–17, several large-scale studies, systematic reviews and meta-analyzes were carried out, which showed that expression profiles of circulating miRNAs, especially using their combination, have great potential diagnostic value for accurate and early detection of breast tumors [12,13, 14].

In other works, numerous microRNAs that are specific for other types of oncology have also been established, which can help to more effectively detect the disease in its early stages [15,16]. In general, miRNA analysis can be used to diagnose almost all types of this pathology.

But that's not all. It was found that miRNAs can also be effectively used in the diagnosis of the main age-related neurodegenerative pathologies, the progress in the treatment of which today is weak.

So, in 2015, a meta-analysis of 8 studies was conducted, in which 459 patients with neurodegeneration and 340 healthy people in the control group took part, to study the diagnostic indicators of circulating miRNAs. A meta-analysis confirmed that miRNAs can be potential biomarkers in the clinical diagnosis of neurodegenerative diseases, and their diagnostic accuracy will be better using the analysis of several miRNAs [17]. 

In 2018, a study of American neurologists was published, in which the levels of specific miRNAs in the liquor of people carrying genetic mutations associated with Huntington's disease were studied. As a result, they were able to detect 6 microRNAs (miR-520f-3p, miR-135b-3p, miR-4317, miR-3928-5p, miR-8082, miR-140-5p), whose levels began to show growth 20 years before expected appearance of the first symptoms of the disease. That, according to scientists, increases the chances of making the treatment effective and postponing the onset of the disease [18].

In other studies, specific miRNAs (miR-455-3p, miR-501-3p, miR-26a-5p, miR-181c-3p, miR-126-5p, miR-22-3p, miR-148b-5p, miR-106b-3p, miR-6119-5p, miR-1246, miR-660-5p)) allowing more accurate and early diagnosis of another severe neuropathology - Alzheimer's disease [19,20,21].

Also, studies have shown that miRNAs have the potential to be used not only in diagnosis, but also as therapeutic targets in the treatment of diseases [22, 23]. And this is not surprising. According to modern estimates, the expression of about 60% of human genes is directly related to the action of miRNA, and the functions of most of them remain unclear [24].

MicroRNAs have shown themselves to be very sensitive biomarkers, allowing to detect the disease on the most distant approaches, when no symptoms and pathological changes are visible. What makes them a unique tool in the fight for healthy longevity. And although with respect to miRNA one can still come across the definition of “dark matter of biology,” already what is known now shows great prospects for studying these small molecules.

Review author: Alexey Rzheshevsky.