The role of Exosomal miRNAs in cancer

Reports assessing exosomes in relation to cancer biology have been on an exponential rise recently. Exosomes represent membrane-bound vesicles with diameters ranging between 30 and 150 nm, and are found in virtually all body fluids. Most cell types release these molecules into the extracellular space upon fusion with the cytoplasmic membrane [1]. Exosomal content involves unique miRNAs, mRNAs, DNA fragments, lipids and proteins, which genetically specify their cells of origin [2]. Exosomes are then taken up by other cells for subsequent modulation of recipient cells. Currently, miRNAs in exosomes are attracting increasing attention due to their contributions in recruiting and reprogramming important components of the tumor microenvironment. They are also considered to be essential in intercellular communication along with cell-to-cell contact-related signaling through the transfer of soluble molecules [3]. This represents an emerging concept, with a shallow understanding of exosomal miRNAs in general. The current review examined the biological properties of exosomes, discussed the proposed mechanisms of miRNA loading into exosomes, highlighted the mechanisms by which exosomal miRNAs affect tumor progression and outlined the potential use of exosomal miRNAs as biomarkers in multiple malignancies [4].

At present, exosomes have been found to be mainly generated by mechanisms dependent on or independent of endosomal sorting complex required for transport (ESCRT).ESCRT consists of different protein complexes and helper proteins, which participate in the formation of membrane-invagination and polyvesicle body (MVBs), and mediates the fusion of MVBs with cell membrane, resulting in the formation of exosomes. Meanwhile, IT has been found that MVBs can also be produced in a mechanism independent of ESCRT, enriching the research on exosome formation [5].

The biogenetic process leading to exosomal miRNAs starts in the nuclear compartment, in which DNA sequences producing miRNAs undergo transcription by RNA polymerase for generating primary miRNAs (pri-miRNAs). The generated pri-miRNAs are initially included in longer molecules, and undergo processing in the nucleus to form 70–100 nt long hairpin RNAs [6]^. Exportin 5 assists in the transportation of hairpin pri-miRNAs to the cytoplasm, in which they are further processed by Dicer. Upon maturation, these double-stranded miRNAs are changed into single-stranded miRNAs, followed by miRNA sorting into exosomes. Based on the current research, it has been deduced that miRNA incorporation into exosomes is not a random event. Although the underlying mechanisms require clear understanding, the cells of origin use sorting processes that specifically guide intracellular miRNAs into exosomes [7]. There are about five potential channels performing miRNA sorting into exosomes, which are described below. (1) Neural sphingomyelinase 2 (nSMase2)-related pathway: nsMase2 was the first protein reported to be related to miRNA secretion into exosomes. Its downregulation decreased exosomal miRNA amounts, while its overexpression increased exosomal miRNA levels [8]. (2) The pathway associated with the 3′ ends of miRNAs. The 3′ ends of endogenous miRNAs containing uridines are mostly present in B cell- or urine-derived exosomes [9]. Therefore, the 3′ ends of miRNAs contain a powerful sorting signal. (3) The pathway associated with miRNA motifs and sumoylated heterogeneous nuclear ribonucleoproteins (hnRNPs). Sumoylated hnRNPA2B1 recognizes the GGAG motif at the 3′ end of miRNAs, causing the packaging of specific miRNAs into exosomes [10]. HnRNPA1 and hnRNPC are the other two hnRNP family members also binding to exosomal miRNAs, indicating their involvement in miRNA sorting. (4) MiRNA induced silencing complex (miRISC)-related pathway. Here, mature miRNAs and assembly proteins form the miRNAISC complex, which mainly contains miRNAs, miRNA-repressible mRNAs, and GW182 and AGO2 (Argonaute-binding proteins). The binding of mRNAs by these miRNAs occurs mostly within their 3′ untranslated regions (3′UTRs) [11]. The mRNA/miRNA complex next suppresses translation by preventing initiation or increasing mRNA degradation. Recently published findings have demonstrated a potential association of AGO2 with exosomal miRNA sorting. Data have indicated AGO2 knockout suppresses miRNAs such as miRNA-150, miRNA-142-3p and miRNA-451 in HEK293T-derived exosomes [10]. (5) Ceramide related pathway: As an exogenous ceramide supplement, ceramide C6(C6-cer) can dose-dependent inhibit the proliferation of human multiple myeloma (MM) cells and promote cell apoptosis Further studies showed that C6-cer treatment increased the levels of some tumor-suppressive mirnas (e.g. miR202,miR16,miR29b and miR15a) in exosomes, and the levels of these tumor-suppressive mirnas were also exosomes Changes also occurred in C6-cer treated MM cells, suggesting that the ceramide pathway plays an important role in miRNA incorporation into exosomes [12]. After miRNAs enter the exosomes, many reports have proposed mechanisms by which exosomes are secreted outside the cells, so this is not discussed in this review. The biogenetic and sorting mechanisms of exosomal miRNAs are presented in Fig. 1.

Exosome miRNAs can be detected by a variety of methods, including quantitative reverse transcription polymerase chain reaction (qRT-PCR) 、fluorescence and ratio-based electrochemistry Local surface plasmon resonance (LSPR) quantitative methods for exosome miRNA without PCR, these methods enrich the detection technology of exosome miRNA to a certain extent, but there are still some problems such as poor RNA stability, complex and expensive technology and false positive signal [13]. In addition, precision medicine has made great progress in the medical field. The detection of circulating miRNA(C-miRNA) as part of precision medicine has been paid attention to C-miRNA as circulating targets (CTs) is readily available in a variety of body fluids and is released into body fluids in the form of miRNA protein complexes or EV-shuttle complexes, ensuring the high stability of C-miRNA during circulation and repeated freeze–thaw in long-term storage has been reported C-miRNA levels are stable under heating or extreme pH conditions, and thus can be used as a reliable CTs for liquid biopsy. Abnormal expression of C-miRNA can distinguish healthy patients from disease patients. In addition, C-miRNA helps to identify tumor origin and classify tumor types and subtypes, which are important for cancer diagnosis, treatment and prognosis Similarly, the detection of C-miRNA still has some problems, such as expensive equipment and complex operation, which still needs further exploration [14].

Diagnosing cancer at an early stage could reduce disease-related deaths and extend the lifespan. Exosomes have become a hot research topic as a biomarker for diagnosing multiple diseases. Previous researches have revealed that miRNAs enclosed in exosomes could escape immune attacks and cross the blood–brain barrier. In addition, exosomes prevents the clearance and/or damage of miRNAs by complement fixation and macrophages because of their double-layered membrane and size in the nanometer range, prolonging their circulatory half-life and improving clinical diagnosis. Mounting evidence indicates exosomal miRNAs are circulating diagnostic markers for different types of cancer [108]. The unique features of miRNAs in exosomes could be modified to increase their diagnostic efficacy for use as ideal tools for non-invasive screening and targeting of cancer cells in clinic. Several reviews discussing serum exosomal miRNAs in multiple malignancies are available. However, utilizing exosomes and their cargo as a diagnostic and monitoring tool for diseases remains challenging, requiring further investigation. Meanwhile, accurate purification, identification and high-throughput use of extracellular vesicles in clinic face enormous challenges. Further developing tumor exosomal miRNAs and improving microfluidics for exosome detection could improve their use for diagnosing cancer.