All organisms transmit genetic information from parent to offspring through vertical gene transfer. Bacteria also have horizontal (lateral) gene transfer for exchanging genetic information, which allows them to diversify their populations and facilitate adaptation to new conditions. Horizontal information transfer in eukaryotes was discovered relatively recently through the intercellular transport of miRNAs [39]. In 2008, circulating miRNA (c-microRNA) was first detected in maternal plasma [40]. Later, c-microRNAs were also found in other biological fluids, such as blood (serum, plasma), urine, cerebrospinal fluid, saliva, milk, lacrimal and seminal fluids, and bronchial lavage [41,42,43]. The appearance of c-microRNAs in the blood can result from secretion by cells or from cell death during apoptosis, necrosis, tumors, or trauma.

Interestingly, despite the presence of various nucleases, circulating endogenous miRNAs remain stable while pure exogenous miRNAs added to plasma degrade rapidly [41]. Subsequent studies established that endogenous miRNAs are highly stable and resistant not only to endogenous ribonucleases but also to extreme temperatures, pH levels, and freeze–thaw cycles [44]. To ascertain what protects them from enzymatic degradation, let us consider the mechanisms of miRNA entry into extracellular fluids and the forms in which they are found there. Initially, c-miRNAs can be secreted into the extracellular space in microvesicles (late endosomal compartments [45]), ectosomes [46], exosomes [47], or microvesicles (liposomes), or they can be associated with high-density lipoproteins (HDL) [48], or released as parts of apoptotic bodies. However, most of them, according to Reiner et al., form complexes with the proteins AGO2 or NPM1 (nucleophosmin 1) [49]. Regardless of the form in which they enter the extracellular space, miRNAs then pass into other biological fluids, for example, the general bloodstream.

Microvesicles (microparticles or ectosomes) are plasma membrane-derived particles released into the extracellular space by budding out and detachment from the plasma membrane. They range in size from 100 nm to 1 µm and are formed by outward protrusion of the plasma membrane, followed by separation [50]. Ectosomes are secreted by various cells, including tumor cells, polymorphonuclear leukocytes, aging erythrocytes, and activated platelets. One of their characteristic features is the appearance of phosphatidylserine (PS) on their membrane surfaces. Unlike exosomes, ectosomes bind well to annexin V and can also bind to prothrombin and blood coagulation factor X to form the prothrombinase complex [41].

Exosomes are small, membrane-bound vesicles of endosomal origin, 30–100 nm in diameter, that form intracellularly via endocytic invagination and are released into a structure known as the multivesicular body (MVB). The MVB then fuses with the plasma membrane, releasing the exosome contents into the extracellular space [51]. Exosomes are secreted by almost all normal cells (T-cells, B-lymphocytes, dendritic cells, reticulocytes, neurons, intestinal epithelial cells, platelets, etc.) and by pathological cells. It was previously believed that exosomes function only as protein scavengers, but in 2007, Valadi et al. found that they can carry nucleic acids (NAs), in particular, RNA [52]. It is now known that they can contain various components of their donor cells, including proteins (proteolytic proteins, chaperones (Hsp70 and Hsp90), apoptotic proteins (Alix), translation factors, metabolic enzymes), lipids, mRNA, microRNA, small interfering RNA (siRNA), and DNA. They can also carry viruses and prions from an infected cell [53]. Thus, exosomes function as horizontal carriers (between cells of the same organism, donor–recipient) of information, mRNA, viruses, and other materials, such as proteins and microRNAs. Some proteins are exosome-specific and do not depend on the donor cell; however, there are also specific ones, so it is possible to identify a subpopulation of specific exosomes and infer the cell type of origin [54]. However, how the various components making up exosomes are chosen remains an open question. In 2012, 4563 proteins had been found in exosomes [55], and the list of exosomal miRNAs numbered about 800. Cells can interact with each other by transferring exosomes loaded with miRNAs [56,57]. The authors of [57] showed that monocyte exosomes deliver miR-150 to endothelial cells and enhance their migration by reducing c-myb 9 expression. The miRNA content of exosomes is critical in this type of intercellular communication and determines the fate of the recipient cell. Thus, exosomes derived from mesenchymal stromal cells of the bone marrows of myeloma patients promote tumor growth, and this effect depends on their miR-15a content.

Exosomes produced by stressed cells also provide information that induces resistance in surrounding cells through a paracrine effect. For example, miRNAs regulate the activity of pancreatic β-cells by transfer via exosomes [58]. Exosomes are also involved in transmitting the immune response; miRNAs transferred by exosomes from T-cells to antigen-presenting cells (APCs) can regulate gene expression in the recipient cells [59]. Exactly this process accounts for the suppression of antitumor immunity, including inhibition of T-lymphocyte and natural killer activities, and the suppression of APC differentiation. Tumor exosomes also enhance the activity of immunosuppressive cells and increase their number. There are indications that tumor cells dispose of chemotherapy drugs (in particular, doxorubicin) by secreting them in exosomes. This process underlies the acquisition of resistance to anticancer therapy by malignant cells [60]. Considering these properties, exosomes can be considered promising tools for delivering drugs and engineering exogenous miRNAs for high-precision treatment of various diseases. Details about exosomes, their functions, mechanisms of action, and clinical applications have been reviewed [53,61].

In the extracellular fluid, some c-miRNAs are complexed with HDL [24,48]. Cellular export of miRNAs to HDL is regulated by neutral sphingomyelinase. The authors have demonstrated that reduced HDLs injected into mice form complexes with various miRNAs in normal and atherogenic models. The HDL-miRNA profile differs significantly between a healthy person and one with hereditary hypercholesterolemia. It is noteworthy that HDL-miRNAs in atherosclerosis induce differential gene expression with a significant loss of conserved mRNA targets in cultured hepatocytes. Taken together, these observations indicate that HDL is involved in intercellular communication, including the transport and delivery of miRNAs.

Other vesicles that transport miRNAs in the intercellular fluid are apoptotic bodies. These are vesicles with a diameter of 50 nm to 4 μm, formed from cells undergoing apoptosis [62].

The foregoing shows that c-miRNAs function as secreted signaling molecules and affect the phenotypes of recipient cells. Numerous studies have correlated the levels of vesicular and protein-bound c-miRNAs with various pathologies. Moreover, secreted extracellular miRNAs can reflect molecular changes in the cells from which they originate, so their profiles differ depending on physiological and pathological conditions [63]. They can therefore be considered potential diagnostic markers for diabetes, systemic lupus erythematosus, asthma, arthritis, Alzheimer’s disease, cardiovascular diseases, various tumors, etc. [64,65,66].

In 2011, the existence of c-miRNAs in vesicular form was questioned, since only 10% of circulating microRNAs are in this form [67]; the remaining 90% circulate in association with proteins of the Argonauts family (AGO2), HDL, or other RNA-binding proteins [68]. However, it was later found that the non-vesicular forms of c-miRNA are non-specific products of physiological activity and cell death.

The basis on which one method of miRNA transport is chosen over another for delivery into the intercellular space is not clear. However, selective “packaging” of various c-miRNAs into microvesicles and exosomes has been demonstrated [68,69,70]. For example, the let-7 miRNA family in a metastatic gastric cancer line is selectively secreted into the extracellular environment exclusively by exosomes. According to the authors, this contributes to the maintenance of oncogenesis and metastasis [68]. Wang et al. showed that some human cell lines (HepG2, A549, T98, and BSEA2B) actively release miRNAs for an hour immediately after serum deprivation [71], suggesting that miRNAs are secreted in response to stress. In this experiment, the authors noticed that most of the extracellular c-miRNAs were complexed with RNA-binding proteins, not inside microvesicles or exosomes. Characteristic miRNA sequences promote interaction with the A2B1 fish nucleoprotein, while Ago2 facilitates miRNA loading into the vesicle. A mechanism for miRNA sorting based on the 3′-terminal structure was proposed [72]. These authors found that 3′-terminal-adenylated microRNAs were predominantly intracellular, while their 3′-terminal-uridylated isoforms were found in exosomes. This confirms the influence of post-transcriptional modifications on the microRNA sorting method.

miRNA загружается в везикулу за счет взаимодействия характерных мотивов miRNA с нуклеопротеином рыбы A2B1, что облегчает процесс. Интересно, что количество микроРНК, избирательно высвобождаемых из клеток в конкретную жидкость организма, может коррелировать со злокачественными новообразованиями [ 73 ]. Пигати и др. обнаружили, что большая часть miR-451 и miR-1246, продуцируемых злокачественными эпителиальными клетками молочной железы, высвобождается во внеклеточное пространство, в то время как большая часть тех же miRNAs, продуцируемых нормальными эпителиальными клетками молочной железы, остается в клетке. Эти результаты подтверждают механизм клеточного отбора для высвобождения miRNA и указывают на различия между их внеклеточным и клеточным профилями. Такое селективное высвобождение позволяет рассматривать c-miRNAs как биомаркеры различных заболеваний.

Механизмы, которые регулируют и контролируют высвобождение экзосом в клетку-реципиент, остаются совершенно неизвестными. Однако идентифицированы некоторые белки, участвующие в этом процессе: ТАТ-5 и Rab27. TAT-5 представляет собой белок, ассоциированный с клеточной мембраной, который регулирует отделение пузырьков [ 74 ]. Rab27 участвует в экзосомальном высвобождении и поглощении реципиентными клетками [ 75 ]. Высвобождению экзосом из клеток млекопитающих также способствует церамидный путь, который также ингибирует экспорт miRNAs в сочетании с HDL.

3. Преимущества и недостатки микроРНК как предиктора заболевания

4. Использование микроРНК в качестве мишеней при лечении заболеваний

5. Целевые стратегии доставки микроРНК в естественных условиях