Expression, regulation, and function of exosome‐derived miRNAs in cancer progression and therapy

Exosomes are a novel class of intercellular signal modulators that contain a wide range of molecules and deliver information between cells and tissues. MicroRNAs (miRNAs), a type of regulatory non‐coding RNA, are often incorporated into exosomes as signaling molecules. In this review, we discuss the expression of exosomal miRNAs from diverse origins such as tumor cells, solid tumor tissue, and biological fluids in various cancers (lung, breast, colorectal, liver, stomach, and pancreatic). We address the biological functions of exosome‐derived miRNAs in processes such as tumor‐cell proliferation, angiogenesis, metastasis, and chemoresistance in the tumor microenvironment. In particular, we discuss three oncogenic miRNAs, miR‐21, miR‐141, and miR‐451, which occur within exosomes, in terms of gene regulation and intercellular communication. We consider therapeutic miRNA‐based nanoparticles, which are widely expressed in tumors and show promise in drug therapy. The review assesses the wide‐ranging evidence for using exosomal miRNAs as tumor markers in molecular diagnosis. Further, we consider the use of nanoparticle platforms to transport miRNAs, in the targeted treatment of disease and tumors.


| INTRODUCTION
Most cancers cause great harm to public health, because of their high mortality and poor prognosis. The GLOBOCAN 2020 database provides the latest global cancer burden. In 2020, an estimated 19.3 million new cancer cases and almost 10.0 million cancer deaths occurred worldwide. The global cancer burden is expected to be 28.4 million cases in 2040. 1 Early detection of malignancies can improve prognosis and survival. 2 Although biopsy is the main method to diagnose tumor progression and metastasis, 3 more advanced and safer methods with high sensitivity and specificity are needed in cancer diagnosis. Exosomes are widespread in various body fluids, including blood and urine. Because of their heterogeneous contents, they can provide information about their cells or tissues of origin; this information can be used in disease diagnosis. In addition, they are internalized by specific cell types, thereby transmitting their contents and promoting cell communication. Exosomes can be loaded with drugs, acting as carriers and permitting novel treatment strategies. The novel use of exosomes for tumor diagnosis has attracted attention. 4 Determining the intrinsic connections between exosomal miRNAs expression and cancer progression has substantial therapeutic and monitoring significance. Hence, this review focuses on the characteristics of exosomal miRNAs in various types of cancer, outlines the common and distinctive features of cancer-exosome-derived miRNAs, highlights miRNA-associated tumor-promoting and tumor-suppressing signals and pathways, and explores the mechanisms of exosomal miRNA involvement in tumor biology.

| DEFINITION, COMPOSITION, AND FUNCTION OF EXOSOMES
In 1981, researchers observed that exfoliated cells from cultures of cell monolayers showed ecto-ATPase and ecto-5′ exonuclease activity. 5 These particles, named exosomes by Johnstone et al., 6 are small cup-shaped membranous particles (diameter 30-150 nm) encapsulated by a lipid bilayer. They are released into extracellular spaces by cells when intracellular multivesicular bodies fuse with the cell membrane during normal physiological and pathological processes. 7 Although many mechanisms of exosome biogenesis have been revealed, the most characteristic mechanism is via the endosomal sorting complex required for transport (ESCRT) pathway. 8 First, ESCRT-0 recognizes ubiquitous shutters and promotes the initiation of exosome budding; ESCRT-0 then recruits ESCRT-I, which recruits ESCRT-II, which may play a key role in cargo clustering; ESCRT-III then disassembles ESCRT-0, ESCRT-I, and ESCRT-II to promote exosome budding, via Vps-4. 9 Exosomes exist in various biological fluids, such as urine, 10 cerebrospinal fluid, 11 bronchial lavage, 12 ascites, 13 breast milk, 14 saliva, 15 serum, 16 and plasma. 17 Various cells, including lymphocytes, macrophages, mast cells, adipocytes, and tumor cells, can secrete exosomes. 18 Although exosome composition differs between cell types, they comprise mainly proteins, nucleic acids, and lipids. 9 Researchers have identified 9769 proteins, 3408 mRNAs, 2838 miRNAs, and 1116 lipids within exosomes. 19 Exosomal proteins are mainly cargo or membrane proteins, and participate in processes such as antigen presentation, membrane transport and fusion, and immune defence. 9,19 Lipids, critical components of the exosome membrane, participate in many processes and stabilize exosome contents, allowing exosomes to be used as biomarkers and drug-delivery vehicles. 20 Exosomes can be modulated by donor cells and exchange or transmit information to recipient cells in healthy tissue. 21 Under pathological conditions, however, exosomes can influence disease progression. For example, they promote angiogenesis in hepatocellular carcinoma (HCC) and lung cancer and promote metastasis. 9 Cancercell-derived exosomes transmit 14-3-3ζ from HCC cells to T cells, thereby inhibiting the anti-tumor function of tumor-infiltrating T cells. 22 Kras G12D -targeting iExosomes, exosomes with short interfering RNAs or short hairpin RNA, can significantly reduce oncogenic Kras G12D mRNA levels and suppress Kras G12D -expressing human pancreatic orthotopic tumors; they can also inhibit tumor metastasis and increase overall survival. 23 Moreover, exosomes can influence human immune responses: for instance, premetastatic tumor-derived exosomes induce a patrolling monocyte-dependent innate immune response, by mediating immune surveillance, thus eliminating cancer cells in the premetastatic microenvironment. 24

| miRNAs and oncogenic miRNAs (oncomiRs)
Genomic analyses have revealed that only 2% of RNA encodes proteins; therefore, many non-coding genome regions are transcribed into non-coding RNAs (nc-RNAs). miRNAs are a family of regulatory ncRNAs. 25 miRNAs are single-stranded RNAs comprising 21 to 24 nucleotides and have broad regulatory functions. 26 In 1993, Ambros and co-workers identified lin-4 in Caenorhabditis elegans; this was the first miRNA identified; further, such miRNAs did not encode a protein, but regulated other RNAs. 27 The latest miRBase database (v. 22, March 2018) contains 38 589 entries from 271 organisms, and uncovered 2654 mature miRNAs in humans. 28 miRNAs participate in regulating cell development, differentiation, proliferation, cell death, and metabolism. In addition, miRNAs are related to various pathological states, such as those associated with infectious disease and carcinoma. 3,29 In humans, >60% of protein-coding genes are regulated by miRNAs. 30 Cancer-related miRNAs that influence tumorigenesis and development, functioning as tumor suppressors and oncogenes, are known as on-comiRs. OncomiRs overexpression, or low expression of tumor-suppressing miRNAs, is related to human cancers. 31 This suggests that miRNAs influence most biological processes in humans. Based on this evidence, miRNAs are key regulators of various tumor processes and have the potential to act as specific and sensitive biomarkers for cancer.

| Exosomal miRs as potential tumor biomarkers
Many studies have shown that exosome-derived miRNAs may be more powerful than mRNAs or proteins as cancer biomarkers because exosomal miRNAs contribute to tumorigenesis, prognosis, and responsiveness to therapy. For instance, serum exosomal miR-1247-3p levels are associated with lung metastasis in patients with HCC. 32 Serum exosomal miR-210 originating from tumor tissue is associated with tumorigenesis in clear cell renal cell carcinoma. 33 Circulating exosomal miR-21 levels are closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC). 34 In a prostate cancer (PCa) metastasis model, risk-scoring based on urinary exosomal miR-21, miR-451, and miR-636 performed better than that based on preoperative prostate-specific antigens. 35 Therefore, exosomal miRNA expression has potential indicative value in tumor diagnosis.
Research has focused on the possible relationships between exosomal miRNAs and tumor types, to discover novel tumor-specific and sensitive cancer biomarkers. For instance, miR-21 and miR-1246 are selectively enriched in exosomes and are significantly elevated in the plasma of patients with breast cancer. 36 Circulating exosomal miR-106a-5p and miR-20b-5p, from the miR-106a-363 cluster, are consistently upregulated in breast cancer tissue. 37 Higher serum expression of exosomal miR-19a indicates poorer colorectal cancer (CRC) prognoses. 38 Higher circulating levels of exosomal miRNA-21 and lncRNA-ATB are independent predictors of mortality and disease progression in HCC. 39 Higher plasma exosomal miR-1290 and miR-375 levels are markedly associated with poor overall survival in castration-resistant PCa. 40 After radiotherapy treatment, serum-derived exosomal hsa-let-7a-5p and hsa-miR-21-5p were elevated in patients with PCa with high-risk disease. 41 However, reduced exosomal miR-548c-5p levels predict poor prognosis in CRC. 42 These findings reveal the potential value of exosomal miRNAs as novel non-invasive biomarkers for tumor detection and prognosis.

| Exosomal oncomiRs in tumor proliferation
Exosomal miRNAs participate in tumor proliferation and tumorigenesis. For instance, breast-cancer-secreted exosomal miR-105 is captured by cancer-associated fibroblasts (CAFs); metabolic reprogramming of stromal cells, in turn, contributes to sustained tumor growth by conditioning the shared metabolic environment. 43 Plasma exosomal miR-19b-3p from patients with esophageal squamous cell carcinoma (ESCC) suppresses MAP2K3 expression to promote ESCC-cell proliferation. 44 In vitro coculture of miR-155-rich or miR-21-rich exosomes promote OSCC-cell proliferation and invasion by downregulating PTEN and Bcl-6. 45 Exosomal miRNAs suppress tumor-cell proliferation and metastasis. CAF-derived exosomal miR-34a-5p can be transferred to OSCC cells, binding to AXL, the direct downstream target of CAF, thereby suppressing OSCC-cell proliferation and metastasis. 46 HCC cells can internalize stellate-cell-derived exosomes loaded with miR-335-5p, leading to upregulation of miR-335-5p in cancer cells, thus inhibiting HCC growth and invasion. 47 Likewise, it has been suggested that CAF-mediated HCC tumorigenesis is partially associated with the loss of antitumor miR-320a in CAF exosomes. 48

| Exosomal oncomiRs in tumor angiogenesis
Angiogenesis, a pivotal element in the progression of malignancy, is responsible for the rapid development, early invasion, and poor prognosis of cancer. 49 Tumorassociated exosomal miRNAs participate in tumor angiogenesis and vascular permeability. For instance, miR-23 is highly enriched in metastatic or premetastatic nasopharyngeal carcinoma (NPC) tissue; overexpression of exosomal miR-23a in NPC promotes angiogenesis by suppressing testis-specific gene antigen (TSGA10). 50 Likewise, tumor angiogenesis may be promoted by targeting SMAD4 and STAT6, by transferring HCC-cell-derived exosomal miR-210 into endothelial cells. 51 The A549 lung cancer cell line delivers miR-494 into vascular endothelial cells via an exosome-mediated route, promoting angiogenesis by targeting PTEN and subsequently activating the Akt/eNOS pathway. 52 Moreover, CRC-derived exosomal miR-25-3p alters the expression of vascular endothelial growth factor receptor 2, ZO-1, occludin, and claudin5 in endothelial cells by targeting KLF2 and KLF4, thereby potentiating vascular permeability and angiogenesis. 53 Colorectal-cancer-derived exosomal miR-21-5p can be delivered to endothelial cells and promote angiogenesis and vascular permeability by targeting KRIT1. 54

| Exosomal oncomiRs in tumor chemoresistance
Accumulating evidence indicates that exosomes can facilitate chemoresistance within the tumor microenvironment. For instance, MDEs transmit miR-365 selectively to pancreatic ductal adenocarcinoma (PDAC) cells, hence inducing gemcitabine resistance in PDAC cells. 63 M2 macrophages mediate cisplatin resistance by delivering exosomal miR-21 to gastric cancer cells. 64 miR-223-enriched exosomes released from macrophages are transferred to epithelial ovarian cancer (EOC) cells, hastening induction of chemoresistance in EOC cells. 65 Paclitaxel-resistant ovarian cancer exosomes contain higher levels of miR-1246 than their paclitaxel-sensitive counterparts, and anti-miR1246 treatment significantly sensitizes ovarian cancer cells to paclitaxel. 66 CAFs carrying exosomal miR-196 confer cisplatin resistance in head and neck cancer (HNC) by targeting CDKN1B and ING5. 67 In contrast, some exomiRs are emerging as novel therapeutic targets with anti-tumor effects. For example, temozolomide (TMZ)-resistant glioblastoma multiforme (GBM) cells transmit chemoresistance to recipient TMZsensitive cells in an exosomal miR-151a loss-dependent manner. 68 ExomiR-122 derived from adipose tissue mesenchymal stem cells (AMSCs) can be used as an effective messenger to mediate communication between AMSCs and HCC cells, to render cancer cells sensitive to chemotherapeutic agents, by impacting miR-122 target gene expression in HCC cells. 69 MiR-128-3p delivery via exosomes mediates the increased chemosensitivity of oxaliplatinresistant CRC 70 (Figure 1).

| miR-21
miR-21, one of the first miRNAs detected in the human genome, is located on chromosome 17q23.1. 71,72 Functional experiments showing that it is an oncomiR have revealed that its overexpression is associated with oncogenesis in many forms of cancer. 73 The primary mechanism of miR-21-mediated oncogenesis involves inhibiting the expression of various downstream target genes, such as PTEN [74][75][76] and programmed cell death 4 (PDCD4), via direct binding to the 3′-UTR of target transcripts. 77 Recent studies have confirmed that novel genes, including sprouty 2, 78 ten-eleven translocation 1, 79 protein phosphatase 2 regulatory subunit B alpha, 80 cell adhesion molecule 2, 81 dual specificity phosphatase 8, 82 and Ras association domaincontaining protein 8, are targeted by miR-21 in various tumors and participate in tumorigenesis.
It is possible that miR-21 abundance is regulated by lncRNAs that competitively bind miRNAs as competitive endogenous RNAs, thereby affecting the regulation of downstream target genes. For instance, forced expression of the lncRNA MEG3 (maternally expressed gene) reverses miR-21-mediated activation of the PI3K/Akt pathway in breast cancer cells. 83 Likewise, LINC00312 regulates CRCcell malignancy by binding to PTEN-targeting miR-21. 84 miR-21 overexpression inhibits PTEN and reverses the effect of lncRNA growth arrest-specific 5, resulting in increased proliferation, migration, invasion, and EMT of OSCC cells via the PI3K/Akt pathway. 85 Conversely, miR-21 levels are upregulated by oncogenes such as KRAS 86 and CBX7, 87 leading to tumor onset and development. KRAS transactivates miR-21 and miR-30c via downstream activation and recruitment of ELK1 to the proximal promoters of miRNAs. 86 CBX7, a constituent of polycomb repressive complex 1, upregulates miR-21 by activating the AKT-NF-κB pathway, thereby contributing to the CBX7-mediated stem cell-like characteristics of gastric cancer cells. 87 miR-21 levels were increased by EGF, thereby promoting EGF-induced PDAC cell proliferation. 78 It has been validated that exosomal miR-21 influences tumor-cell proliferation, migration, and invasion. For instance, miR-21-enriched exosomes enhance PTENp1promoter methylation by targeting TETs, thus inhibiting PTENp1 and PTEN expression, thereby promoting HCC cell growth. 88 Exosomal miR-21-5p induces peritoneal mesothelial cell MMT and promotes peritoneal tumor metastasis, thereby targeting SMAD7 and thus activating the transforming growth factor-beta/Smad pathway. 60 The EMT transcription factor Snail induces miR-21 expression in human HNC cells, leading to the secretion of miR-21-abundant TEXs to promote the M2 polarization of tumor-associated macrophages. 89 Moreover, a hypoxic microenvironment may cause OSCC to generate miR-21-rich exosomes that are delivered to normoxic cells to promote pro-metastatic behavior. 34 Likewise, hypoxia induces glioma-derived exosomes (GDEs) to express miR-10a and miR-21, to mediate GDE-induced myeloid-derived suppressor cell expansion and activation, by targeting RARrelated orphan receptor alpha and PTEN. 76 Further, an acidic microenvironment induces exosomal miR-21 and miR-10b expression, thus facilitating HCC-cell proliferation, migration, and invasion both in vivo and in vitro 90 (Figure 2). To the best of our knowledge, no tumorsuppression effects have been identified for miR-21.

| miR-451
miR-451a, encoded on chromosome 17q11.2, is abundant in fetal bovine serum. 103 It is considered a valuable biomarker for cancer detection, prognosis, and treatment. miR-451 suppresses various tumor types and participates in reducing target gene expression, thereby preventing tumor-cell proliferation, apoptosis, and even chemoresistance.
Kinesin family member 2A, a microtubule depolymerase that functions in many biological processes, is an independent prognostic factor in lung squamous cell carcinoma; it has been implicated as one of 15 putative F I G U R E 2 miR-21 function and gene regulation pathways. miRNA-21 can be tested in exosomes from serum, plasma, and tumor tissue. miR-21 participates in tumor-cell proliferation, metastasis, and epithelial-mesenchymal transition (EMT) via various downstream molecular pathways, and its expression is regulated by upstream targets. BC, breast cancer; CRC, colorectal cancer; GC, gastric cancer; HNC, head and neck cancer; NSCLC, non-small-cell lung cancer; OSCC, oral squamous cell carcinoma; PDAC, pancreatic ductal adenocarcinoma oncogenic genes regulated by miR-451a. 104 miR-451a can suppress the expression of the endoplasmic reticulum membrane protein B-cell receptor-associated protein 31, which has been identified as a novel cancer/testis antigen, by binding to its 5′-UTR and promoting CRC apoptosis by increasing endoplasmic reticulum stress (ERS). 105,106 miR-451 promotes cellular drug retention in multidrugresistant (MDR) bladder cancer cells (BIU-87/Adr) by reducing the P-gp levels in MDR cells. 107 Likewise, excision repair cross-complementation group 1 protein (ERCC1) is a DNA endonuclease with variable expression in primary tumor specimens; ERCC1 positive tumors are more resistant to cisplatin treatment than ERCC1 negative tumors. miR-451 overexpression selectively enhances the chemosensitivity of ERCC1 high-expression NSCLC cells to cisplatin, by inhibiting PI3K/Akt signaling and downregulating ERCC1 expression. 108 Likewise, miR-451 overexpression directly targets tyrosine3-monooxygenase/ tryptophan5-monooxygenase activation protein zeta (YWHAZ) to inhibit βcatenin expression, in turn elevating breast cancer-cell sensitivity to paclitaxel. Further, intra-tumoral injection of a miR-451 agomir induced tumor suppression in an SKBR3/PR drug-resistant xenograft model. 109 miR-451 overexpression exerted an anti-glioma effect by downregulating Rac1. Knockdown of long stressinduced non-coding transcript 5, which is overexpressed in multiple tumor types, inhibited glioma cell growth and metastasis by upregulating miR-451. 110 miR-451 antagonizes angiogenesis to suppress HCC by directly targeting the interleukin 6 receptor-STAT3-vascular endothelial growth factor pathway 111 (Figure 4).

NANOPARTICLES
Exosomes hold great promise as endogenous nanocarriers that can deliver biological information between cells. Exosomes enriched within metalloproteinase 15 (A15 exosomes), derived from continuous protein kinase C activation in monocyte-derived macrophages, coloaded with doxorubicin hydrochloride and cholesterol-modified miRNA 159 (Cho-miR159) induced synergistic therapeutic effects in MDA-MB-231 BC cells in vitro. 112 In vivo, miR159 and Dox delivery in a vesicular system effectively silenced the TCF-7 gene, producing anticancer effects without adverse effects, in triple-negative breast cancer (TNBC) therapy. 112 Alarmin-painted tumor exosomes, which are used to deliver tumor-associated antigens and the HMGN 1 (N1ND) functional N-terminal domain, strengthened dendritic cell long-lasting anti-tumor immunity and tumor suppression in various syngeneic mouse models with large tumor burdens. 113 Similarly, nanoparticles have been widely used as exogenous delivery vehicles in tumor immunotherapy in vivo and in vitro. For instance, RNA nanoparticles containing an RNA aptamer binding to CD133 and anti-miR-21, F I G U R E 3 miR-141 function and gene regulation pathways. miR-41 acts as a molecular bridge connecting upstream and downstream molecules to promote tumorigenesis. The main effects of exosomal miR-41 on tumorigenesis include tumor-cell proliferation, metastasis, tumor angiogenesis, and chemoresistance carried by a thermodynamically and chemically stable three-way junction motif, were specifically taken up by breast cancer stem cells and TNBC cells. 114 Animal trials in TNBC have demonstrated that systemic injection of RNA nanoparticles promotes highly specific tumor targeting and high inhibition of tumor growth, without inducing cytokine secretion. 114 Ultrasound-targeted microbubble destruction (UTMD)− miRNA combination therapy, which uses UTMD for local delivery of nanoparticle-loaded miRNA-122 and anti-miRNA-21, alters the immune microenvironment in Hepa1-6 tumors by modulating cytokine expression, improving the response of HCC to chemotherapy by eliminating drug resistance. 115 Triple-action nanoparticles acting via CXCR4 antagonism and miR-210/KRAS G12D downregulation improved pancreatic cancer therapeutic effects, as revealed by delayed tumor growth, stromal depletion, reduced immunosuppression, inhibited metastasis, and prolonged survival. 116 In addition to nanoparticle-based delivery, an RNA micelle platform can be used to deliver anti-miRNA agents. 117 An 8 nt LNA-modified anti-miR21 antibody can be annealed to a phi29 DNA-packaging RNA three-way junction (pRNA-3WJ) scaffold via complementation with an extended sequence. Compared to RNA nanoparticles without micelle formation, RNA micelles showed enhanced tumortargeting ability and greater accumulation in tumors, in a mouse xenograft model. 117 siRNAs delivered by lipid nanoparticles or other vehicles require excessively high therapeutic doses, with toxic outcomes. It has recently been shown that the pre-miR-451 backbone can facilitate the enrichment of therapeutic siRNA to the level of the most abundant miRNA in small extracellular vesicles (sEVs), thereby reducing the required delivery volume of sEVs by 100-to 10 000-fold. 118 Therefore, integrating siRNA into the pre-miR-451 backbone offers a powerful and new approach as a delivery platform for anticancer therapy.

| CONCLUSIONS
The detection of cancer biomarkers is significant for diagnosis and prognosis, and fluid/tissue biopsy provides a new approach. 119 Exosomal miRNAs can be extracted from many types of body fluid. They occur in tumor cells, and participate in tumor-microenvironment reactions to promote tumor-cell growth. Serum and plasma analysis of exosomal miRNAs, therefore contributes to early disease detection and treatment monitoring. In therapy, tumor-or immune-cellderived exosomal miRNAs may promote or suppress tumor development, causing drug resistance or promoting tumor invasiveness. Investigating serum and plasma analysis of exosomal miRNAs will facilitate early cancer diagnosis and F I G U R E 4 miR-451 gene regulation pathways have tumor-suppressive effects. miR-451 downregulates many target molecules and regulates known and unknown molecular pathways, thereby inhibiting tumorigenesis treatment monitoring. Exosomal miRNAs-based therapies include impairing oncomiR expression, disturbing cancer development signaling, or potentiating tumor-suppressive pathways to facilitate tumor diagnosis and therapy.

DISCLOSURES
The authors have stated explicitly that there are no conflicts of interest in connection with this article.

AUTHOR CONTRIBUTIONS
Original draft preparation, Bowen Li; Funding acquisition and draft preparation, Yu Cao; designed the outline and revised the manuscript, Mingjun Sun and Hui Feng. All authors have read and agreed to the published version of the manuscript.