Emerging functions and clinical applications of exosomal microRNAs in diseases

Exosomes are an important group of extracellular vesicles that transfer several kinds of biomolecules and facilitate cell-cell communication. The content of exosomes, particularly the amounts of microRNA (miRNAs) inside these vesicles, demonstrates a disease-specific pattern reflecting pathogenic processes and may be employed as a diagnostic and prognostic marker. miRNAs may enter recipient cells through exosomes and generate a RISC complex that can cause degradation of the target mRNAs or block translation of their corresponding proteins. Therefore, exosome-derived miRNAs constitute an important mechanism of gene regulation in recipient cells. The miRNA content of exosomes can be used as an important tool in the detection of diverse disorders, particularly cancers. This research field has an important situation in cancer diagnosis. In addition, exosomal microRNAs offer a great deal of promise in the treatment of human disorders. However, there are still certain challenges to be resolved. The most important challenges are as follow: the detection of exosomal miRNAs should be standardized, exosomal miRNAs-associated studies should be conducted in large number of clinical samples, and experiment settings and detection criteria should be consistent across different labs. The goal of this article is to present an overview of the effects of exosome-derived microRNAs on a variety of diseases, including gastrointestinal, pulmonary, neurological, and cardiovascular diseases, with a particular emphasis on malignancies.


Introduction
Exosomes are membrane-bound extracellular vesicles that are created in the endosomal section of the majority of eukaryotic cells. These vesicles can be detected in biofluids and have important function in physiological processes, particularly in intercellular communication. Being first identified as secreted particles from differentiated reticulocytes [1], exosomes were supposed to be only implicated in waste secretion from the cell for a long time. This changed when the importance of these vesicles in antigen presentation was discovered by Raposo et al. [2]. This observation threw light on the role of exosomes in the transmission of information between cells. In addition to participating in antigen presentation, exosomes have diverse roles in the regulation of immune response, modulation of several biological aspects of tumor cells such as migration and proliferation, and regulation of different types of cell death [3]. A new paradigm in this research area is the importance of exosomes in the transmission of miRNAs to attain intercellular communication [4]. These short-sized non-coding RNAs can go into the recipient cells using exosomes, and establish a RISC complex that can induce degradation of the target mRNAs or inhibit their translation into proteins. Thus, exosome-derived miRNAs constitute an important route of gene regulation in recipient cells. The importance of miRNAs in the pathobiology of human disorders has been reviewed in several studies [5][6][7]. The current review aims to provide an overview of the impact of exosome-derived miRNAs in diverse disorders, including gastrointestinal, pulmonary, neurological, and cardiovascular disorders, with a special focus on cancers.

Exosomal miRNAs in gastrointestinal disorders
The impact of exosome-mediated delivery of miRNAs in the pathogenesis of gastrointestinal disorders has been assessed in malignant and non-malignant conditions (Table 1). In gastric cancer patients, serum exosomal levels of miR-590-5p have been lower in both early (stages I and II) and late (stage III) groups compared with healthy controls. The expression level of this miRNA could differentiate affected individuals from unaffected ones with an area under the receiver operating characteristic curve (AUC) value of 0.810. Most notably, serum levels of exosomal miR-590-5p have been related to clinical stage, infiltration depth, and levels of Ki-67. Moreover, authors have reported a correlation between the down-regulation of exosomal miR-590-5p and a low overall survival rate. In vitro studies have shown that over-expression of miR-590-5p leads to the suppression of cell migration and invasion in gastric cancer cells [8]. miR-122-5p is another downregulated miRNA in serum-derived exosomes of patients with this type of cancer. Exosome-mediated delivery of miR-122-5p could hamper the proliferation and metastatic ability of gastric cancer cells by inhibiting the expression of GIT1 [9]. Conversely, miR-10b-5p has been shown to be over-expressed in tissues and serum exosomes of advanced stages of gastric cancer compared with early-stage samples. Functional studies have shown that miR-10b-5p could target PTEN in gastric cancer cells and KLF11 in fibroblasts. miR-10b-5p silencing up-regulates PTEN levels and repression of PI3K/AKT/mTORC1 signals in gastric cancer cells, resulting in a reduction of colony formation ability and viability of these cells (Fig. 1). In fibroblasts, up-regulation of miR-10b-5p has resulted in down-regulation of KLF11 and elevation of TGFβR1 levels. Taken together, elevated exosomal levels of miR-10b-5p participate in the interactions between gastric cancer cells and fibroblasts in tumor niches by regulating TGF-β signals [10].
In hepatocellular carcinoma, exosomal levels of miR-638 in serum samples have been shown to have a prognostic value through decreasing the expression of VE-cadherin and ZO-1 in endothelial cells [11]. On the other hand, the secretion of exosomal miR-15a from mesenchymal stem cells has been shown to impede the progression of this type of cancer through down-regulating SALL4 levels [12].
In colorectal cancer, several miRNAs have been discovered in cancerderived exosomes that can affect the progression of this type of cancer. For instance, the exosome-mediated transmission of miR-21-5p from colorectal cancer cells to endothelial cells has repressed KRIT1 expression in recipient HUVEC cells and consequently induced activity of β-catenin signals and enhanced expression of VEGF-A and Ccnd1. Totally, these expression changes lead to the enhancement of angiogenesis and vascular permeability. Besides, levels of this miRNA in circulating exosomes have been shown to be elevated in patients with colorectal cancer compared with healthy donors [13]. In addition to the mentioned roles of miRNAs in the pathogenesis of malignant gastrointestinal disorders, exosome-derived miRNAs can affect the pathogenesis of chronic pancreatitis and ulcerative colitis (Table 1).

Exosomal miRNAs in lung cancer
Exosome-mediated delivery of miRNAs is also implicated in the pathogenesis of lung cancer (Table 2). For instance, lung cancer-derived exosomal miR-1260b has been shown to promote the metastatic ability of these cells via the suppression of HIPK2 expression [31]. Moreover, both angiogenic and metastatic abilities and vascular permeability can be enhanced by tumor-originated exosomal miR-3157-3p [32]. In addition, the secretion of miR-155 and miR-196a-5p in the exosomes of tumor-associated macrophages can enhance the metastasis of this type of cancer [33]. On the other hand, exosome-transferred miR-338-3p has a suppressive effect on the metastasis of lung cancer through the inhibition of CHL1 via the MAPK signaling pathway [34]. Exosome-mediated carriage of miR-770 suppresses M2 macrophage polarization by influencing the expression of MAP3K1. This would reduce the invasive abilities of lung cancer cells [35]. Similarly, exosomal miR-3180-3p can inhibit the proliferation and metastatic ability of lung cancer cells through the inhibition of FOXP4 expression [36] (Fig. 2).

Exosomal miRNAs in breast and cervical cancers
Breast and cervical cancers are two types of cancers in which the role of exosomal miRNAs has been investigated (Table 3). Exosomes originating from cancer-associated fibroblasts (CAFs) have been shown to promote the proliferation and metastatic ability of breast cancer cells through transferring miR-500a-5p, a miRNA that inhibits the expression of USP28 [37]. Moreover, these exosomes transfer miR-18b to breast cancer cells to promote their invasion and metastasis via the regulation of TCEAL7 [38]. Meanwhile, the polarization of tumor-associated macrophages can be modulated by the miR-138-5p content of cancer-derived exosomes through the downregulation of KDM6B [39]. On the other hand, exosome-mediated transfer of miR-134-5p can confine the progression of breast cancer by regulating the PI3K/AKT pathway and by influencing the expression of ARHGAP1 [40].
[30] contain miR-1468-5p that induces immune escape through the immunosuppressive reprogramming of lymphatic vessels [41]. Moreover, the delivery of miR-663b by these exosomes enhances the angiogenic ability of cervical cancer cells by inhibiting the expression of vinculin in vascular endothelial cells [42].

Exosomal miRNAs in brain disorders
Exosome-mediated transfer of miRNAs is also implicated in the pathogenesis of glioma, medulloblastoma, traumatic brain injury, neuroinflammation and Rett Syndrome (Table 4). For instance, miR-1246 has been found to be expressed in exosomes extracted from the body fluids of patients with glioma. This miRNA has an important role in the differentiation and activation of myeloid-derived suppressor cells. Exosomal levels of this miRNA in CSF samples after tumor resection have been associated with glioma recurrence rates. Moreover, the expression of miR-1246 in glioma-originated exosomes can be enhanced by hypoxia through a POU5F1 and hnRNPA1-dependent mechanism. Notably, the microtubule inhibitor 2-Methoxyestradiol has been shown to suppress the activation of myeloid-derived suppressor cells through    the inhibition of hypoxia-induced exosomal miR-1246 expression [46]. Plasma levels of another exosomal miRNA, namely miR-2276-5p have been shown to have a potential diagnostic and prognostic role in this type of cancer [47]. Alternatively, the miR-944 content of exosomes originating from glioma stem cells has been shown to reduce the growth and angiogenic ability of glioma cells through the inhibition of AKT/ERK signaling [48]. Finally, the exosomal miR-21-5p content of urine-derived stem cells has been shown to enhance neurogenesis to reduce the progression of Rett syndrome through modulation of the EPHA4/TEK axis [49].

Exosomal miRNAs in cardiovascular disorders
The impact of exosomal miRNAs in the pathogenesis of cardiovascular disorders has been investigated in the contexts of myocardial infarction, thrombosis, atherosclerosis and abdominal aortic aneurysm ( Table 5). miR-143-3p content of exosomes from mesenchymal stem cells has been shown to protect against myocardial ischemia/reperfusion injury through the regulation of autophagy [53]. Moreover, endothelial progenitor cells have been shown to secrete miR-218-5p/miR-363-3p in their exosomes. These miRNAs can amend the pathogenic processes in the course of myocardial infarction through modulation of the p53/JMY signaling pathway [54]. Finally, dendritic cells-originated exosomal miR-494-3p has been found to promote angiogenesis after myocardial infarction [55].
Levels of exosomal miR-145 and miR-885 in serum samples from COVID-19 patients have been significantly correlated with D-Dimer levels. Notably, treatment of human endothelial cells with sera of COVID-19 patients has led to a reduction of miR-145 and miR-885 release, enhancement of apoptosis, and impairment in angiogenic properties. Cumulatively, exosomal miR-145 and miR-885 have been shown to participate in the modulation of thromboembolic events in the context of COVID-19 [56].

Exosomal miRNAs in bone disorders
Exosomal miRNAs have essential roles in the pathogenesis of osteosarcoma, bone metastasis, multiple myeloma, osteoarthritis, postmenopausal osteoporosis, and non-traumatic osteonecrosis of the femoral head (Table 6). Exosomal miR-21-5p originating from bone marrow mesenchymal stem cells has been shown to enhance the proliferation and invasion of osteosarcoma cells via targeting PIK3R1 [62]. Besides, exosomal miR-501-3p originating from osteosarcoma cells can enhance osteoclastogenesis and aggravate bone loss in these patients [63].
Exosomal miRNAs also have an important role in the induction of bone metastasis in estrogen receptor (ER)-positive breast tumors. Exosomal levels of miR-19a and IBSP have been shown to be significantly elevated in bone-tropic ER + breast cancer cells, resulting in the overexpression of these transcripts in the circulation of patients. IBSP can assist in the transfer of exosomal miR-19a to osteoclasts to enhance osteoclastogenesis [64].

Exosomal miRNAs in other disorders
Exosomal miRNAs can also contribute to the pathogenesis of a variety of other malignant (Table 7) and non-malignant disorders ( Table 8). Prostate cancer, oral squamous cell carcinoma, papillary thyroid carcinoma, melanoma, and diffuse large B-cell lymphoma are examples of the former types of disorders, while systemic lupus erythematosus, sepsis, diabetes, diabetic nephropathy, acute and chronic kidney injury, unilateral ureteral obstruction and varicocele are examples of the latter types of disorders.
Suppression of cancer cell-originated exosomal miR-183 has been suggested as an anti-cancer-modality for prostate cancer by affecting the expression of TPM1 [71]. Moreover, exosome-mediated transfer of miR-130b-3p has been shown to promote the progression of oral squamous cell carcinoma and tubular formation via affecting the expression of PTEN [72]. Two miRNAs, namely miR-451a [73] and miR-146a [74] have been shown to contribute to the pathogenesis of systemic lupus erythematosus. Serum levels of the former have been correlated with renal damage [73], and urinary exosomal levels of the latter have been considered as a marker of albuminuria, activity changes, and disease fares in this disorder [74].

Advances and challenges in clinical applications of exosomal microRNAs
Recent studies have shown that exosomal microRNAs serve as vital indicators and disease mediators owing to several characteristics [88][89][90][91]. Exosomes are abundant in numerous bodily fluids and exosomal microRNAs can be collected with reasonable simplicity and little invasiveness [88][89][90][91], making exosomal microRNAs as useful and feasible biomarkers for disease diagnosis. Because exosomal miRNAs are  [77] S. Ghafouri-Fard et al. protected by a lipid bilayer, they are less likely to be degraded by the RNase than free miRNAs [92,93]. This property of exosomal miRNA enables the monitoring of changes in their expression during the course of a disease, as well as the manipulation of disease-related cell signaling in a longer-lasting way [88].

Diagnostic biomarker
Some particular exosomal miRNAs have high diagnostic usefulness in cancers, and their detection aids in early tumor identification. For instance, the signature based on these four microRNAs could successfully differentiate colorectal cancer samples from normal ones [94]. Moreover, serum exosomal miR-134 levels were considerably lower in patients with gastric cancer than in control subjects, and exosomal miR-134 correctly differentiated patients with gastric cancer from matched individuals [95]. In ovarian cancer, miR-1290 was overexpressed in serum exosomes and tissues relative to benign ovarian neoplasm; thus, it may serve as a biomarker for distinguishing ovarian cancer from benign disease [96].

Prognostic biomarker
In addition to their diagnostic value, exosomal miRNAs have been extensively studied in the prognosis of cancers [97]. Downregulation of serum exosomal miR-148a was associated to a worse clinical outcome in breast cancer patients. As a result, exosomal miR-148a in serum may be an important biomarker for breast cancer prognosis [98]. Patients with non-small cell lung cancer who had lower serum exosomal miR-382 levels had a poorer overall survival (OS) rate, suggesting that exosomal miR-382 seems to be an useful predictive biomarker for monitoring the course of non-small cell lung cancer [99]. Plasma exosomal miR-130a levels were significantly higher in 184 patients with oral squamous cell carcinoma than in 196 healthy controls [100]. It was discovered that exosomal miR-130a is an independent predictor of overall survival and recurrence-free survival [100]. Thus, exosomal miR-130a has the potential to serve as a prognostic biomarker in the treatment of oral squamous cell carcinoma. Analysis of 125 patients with colorectal cancer, 70 healthy controls, and 45 benign adenomas revealed that serum exosomal miR-874 was significantly downregulated in colorectal cancer patients [101]. Low serum levels Expression of exosomal miR-874 was related to distant metastatic positivity, lymph node metastasis positivity, poor differentiation, advanced TNM stage, and worse survival [101]. Therefore, exosomal miR-874 serum expression may serve as a reliable colon cancer prognostic marker.

Therapeutic target
Exosomal microRNAs are rapidly being explored as a possible technique for treating cancers. On the basis that exosomal miRNAs effectively attach to target mRNA and decrease gene expression in recipient Hypoxic ucMSC-secreted exosomal miR-125b via targeting TP53INP1 could promote endothelial cell survival and migration in the process of wound healing. [86] cells, malignancies have been treated with tumor suppressor exosomal miRNAs utilizing exosomal engineering techniques [89]. By blocking the MNK/eIF4E axis, exosome-mediated transportation of miR-7-5p improves the anti-cancer effect of Everolimus in non-small cell lung cancer [102]. In addition, exosomes carrying miR-34a promote apoptosis and restrict the migration and development of colorectal cancer cells [103]. Exosomal miR-499 inhibited tumor formation and angiogenesis [104] in endometrial cancer patients, where miR-499 expression was significantly decreased in cancer tissues compared to surrounding tissues. Exosomes that co-deliver 5-fluorouracil and miR-21 inhibitors are able to overcome the 5-fluorouracil (FU) resistance of colon cancer cells and significantly boost their toxicity [105]. miR-34c is a tumor suppressor miRNA that diminishes both malignant behavior and radioresistance in nasopharyngeal cancer [106]. Moreover, exosomes that overexpress miR-34c inhibit tumor formation and enhance the effectiveness of radiation treatment [106].

Challenges in clinical applications of exosomal microRNAs
Exosomal miRNAs offer a great deal of promise, however, there are still certain challenges to be resolved. For example, the detection of exosomal miRNAs should be standardized [107]. Until now, most exosomal miRNAs-associated studies have only been conducted with just a small number of clinical samples used, and experiment settings and detection criteria vary from lab to lab [107]. Additionally, no standardized techniques exist for collecting exosomes, deconstructing them, extracting, and storing miRNA [107]. Furthermore, the vast majority of investigations focused on exosomal miRNA levels in serum and plasma. In contrast, exosomes can be found in a range of physiological fluids, including saliva, tears, and urine [108]. Therefore, before exosomal miRNAs may be utilized as a diagnostic tool in clinical testing, more research and testing will be required to broaden liquid biopsy to uncommon sample sources.
The clinical use of exosomal miRNAs in future cancer treatment has faced some challenges [109]. The effectiveness of exosome-mediated treatment largely relies on exosome source, loading technique, and cell uptake [110]. The first difficulty is producing enough exosomes for clinical trials on a large scale. Bioreactors, 3D scaffolds, and microfluidic devices are used to increase the amount of exosomes [110]. However and quality should be assured while output is going up, especially since exosomes and other kinds of extracellular vesicles can be contaminated or have the same size [110]. The second difficulty is to discover new innovative ways for effectively loading nucleic acids into exosomes, since the poor loading efficiency of existing exosome-nucleic acid loading procedures limits their use [111]. The third challenge is to create precise cancer therapies that are personalized to each individual. The variety of exosomes and the complexities of the in vivo environment make it difficult to predict how and how successfully exosomal miRNA-based therapies will function [112].

Conclusions
Exosome-mediated delivery of miRNAs is implicated in the pathogenesis of a wide array of human disorders. Moreover, the miRNA content of exosomes can be used as an important tool in the detection of diverse disorders, particularly cancers. This research field has an important situation in cancer diagnosis, since cancer cells have been found to secrete higher quantities of exosomes compared with normal cells, and cancer-originated exosomes have a crucial impact on intercellular communications via transporting a variety of growth factors, chemokines, and miRNAs [87], the latter being the focus of this review. The data presented above indicate distinctive expression profiles of miRNAs in the exosomes originating from distinct cellular origins. This finding further highlights the possible application of these vesicles in diagnostic and prognostic approaches. miRNA-loaded exosomes are released not only from cancer cells but also from various immune and mesenchymal cells in the tumor niche, thus they exert diverse roles in cancer biology and affect different aspects of tumor progression, including proliferation, migration, and metabolic states of cancer cells. The content of these exosomes may also affect the response of patients to diverse therapeutic options, including both conventional and targeted therapies. Therefore, these vesicles can be used for establishing personalized routes of cancer treatment. In addition to malignant conditions, exosomal miRNAs affect the pathogenesis of a variety of non-malignant disorders, including immune-related ones such as systemic lupus erythematosus, degenerative disorders, and neurological disorders. Notably, exosomes contain several compounds that may have synergistic effects on recipient cells. Thus, a comprehensive evaluation of exosome cargo is required to determine the precise mechanisms behind their cellular influence. This research field would benefit from the application of novel strategies for exosome isolation and high-throughput sequencing methods for the identification of several targets which are affected by these vesicles.

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Availability of data and materials
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.