Exosomal circRNAs: biogenesis, effect and application in human diseases

Notably, it is increasingly evident that exosomes play an important role not only on the regulation of normal physiological status, such as tissue regeneration [34], immune surveillance [35], blood circulation [36] and stem cell plasticity [37], but also in the pathological conditions in several diseases. For example, exosomes take participate in tissue repair via delivering specific cargoes to the recipient cells. They can enhance the function of regulatory T cells and suppress the CD8⁺ cell and natural killer (NK) activity. In the blood circulation, exosome also take part in breast cancer with lung metastasis from docking Nano platform [38]. Exosomes have been shown in converting the hematopoietic stem cells (HSCs) phenotype into liver cells phenotype [39].

Apart from their fundamental roles in physiological condition, exosomes have a pivotal role in disease pathogenesis, especially in tumor progression. For example, the activated PMN (polymorphonuclear leukocyte) exosomes could bind and degrade extracellular matrix (ECM) by the integrin Mac-1 and NE, respectively, inducing the disorders of chronic obstructive pulmonary disease (COPD) and bronchopulmonary dysplasis (BPD) [40]. A key study showing the clinical relevance and linkage of exosome to disease was reported in 2012 by Wan J and colleagues. This study represented that exosome core component gene was responsible for pontocerebellar hypoplasia and spinal motor neuron degeneration.

Some circRNAs are extraordinarily enriched in the mammalian brain [26] and preferentially back-spliced in neuronal cell lines [41], suggesting that circRNAs have a potential to regulate synaptic function. Similarly, many circRNAs are significantly enriched in synaptogenesis than their linear isoforms during central nervous system aging [42]. In addition, circRNAs are associated with disease pathologic processes. For instance, circRNAs act as microRNA (miRNA) sponges to modulate gene expression. For example, circRNAs are relatively abundant in the brain [26] and accumulate to a high level during the central nervous system aging [42]. Specifically, the circRNA ciRS-7 (also termed CDR1as), which harbors more than 70 conventional miR-7-binding locations, has been observed as a miRNA inhibitor [43]. Additionally, circNT5E acts as a sponge that directly binds miR-422a and inhibits miR-42an activity, regulating multiple pathologic processes, including cell proliferation, migration and invasion in glioblastoma tumorigenesis [14]. Although circRNAs are generally expressed in small quantities, numerous studies have shown that circRNAs are responsible for physiological development and different diseases, such as cardiovascular diseases [44‐47], neurological disorders [20, 48, 49], and tumors [50‐52]. Currently, circRNAs have also been reported to be enriched and stable in saliva [53], plasma [54], and even in serum exosomes [4], suggesting the potential of circRNAs as responsive biomarkers.

Generally, almost all cell types release exosomes that are enriched in plasma as well as other bodily fluids, including saliva, urine, semen, sputum and breast milk. Exosomes are also generated by both immune and nonimmune cells that have a significant role in the regulation of immunity [55]. Moreover, exosome release has been suggested to have prognostic relevance; increased levels of serum exosomes typically predicts unfavorable outcomes [56, 57]. Notably, Li first reported the enrichment and stability of abundant circRNAs in exosomes compared to the producer cells by using RNA-sequence analyses [58]. Although research on exosomal circRNAs is currently gaining momentum, the functions and characteristics of exosomal circRNAs remain largely unclear.

The composition of exosomal circRNAs may be modulated by changes in associated miRNA levels in donor cells, and subsequently this molecular information is transferred to recipient cells. In serum exosomes, circRNAs also contribute to the onset and progression of many neoplasias through RNA-RNA competitive interactions. For instance, circHIPK3 and TUG1 were upregulated, while LncRNA UCA1 was downregulated via RNA silencing, mitogen-activated protein kinase (MAPK) inhibition and in silico analyses [59]. Studies have shown that circRNAs are distinctly downregulated in a KRAS mutant and can be transferred to the exosomes of colorectal cancer cell lines [60]. Currently, exosomal circ-PDE8A was reported to be associated with tumor progression and lymphatic invasion by the miR-338/MACC1/MET pathway in pancreatic ductal adenocarcinoma [61]. Moreover, exosomal circRNA has been observed to promote white adipose browning via targeting the miR-133/PRDM16 pathway, providing a novel perspective into our understanding of cancer-associated cachexia [62]. Additionally, exosomal circRNAs originate from endometrial cancer and hepatocellular carcinoma by targeting the deubiquitination-related USP7, promoting tumor growth [63].

Apart from cancer origin, exosomal circRNAs commonly originate from activated human platelets, which are associated more with hemostasis, inflammation and wound healing than other hematopoietic cells [29, 64, 65]. Interestingly, Zhao showed that exosomal circRNAs might be responsible for the growth and repair of neurons, the transmission of nerve signals, and the regulation of important signaling pathways, specifically in glutamatergic synapses and the cGMP-PKG signaling pathway [66] (Fig. 1). Consequently, further research on exosomal circRNAs not only has profound impacts on understanding the functions and characteristics of exosomes but also provides a novel avenue for many disease diagnoses and targeted Therapies.

Exosome plays a key factor in the formation of premetastatic niches [67] and tumor microenvironment [68]. Additionally, exosomes have been found to associated with tumor cell survival and tumor recurrence through mediating immune suppression and immune surveillance [69, 70]. Interestingly, some circRNAs can be detected in exosomes derived from serum, urine and tumor, exosomal circRNAs may participate in the processes of cell growth, angiogenesis, epithelial mesenchymal transition, and targeted therapy. In this section, we focus on the essential roles of exosomal circRNAs in cell proliferation, tumor metastasis, and drug resistance.

Deregulated proliferation is one of the most important factors in neoplastic transformation; thus, increasing attention has been given to the understanding of the mechanisms of cell cycle regulation [71]. In previous reports, miRNAs were associated with the regulation of the cell cycle and the proliferation of hepatocellular carcinoma (HCC) cells [72, 73]. Interestingly, for arsenite-transformed HaCat cells, Dai reported that exosomal circRNA_100284 regulates the cell cycle by acting as a sponge of miR-217 [74] and inhibits cell proliferation by inducing arrest in the G2/M phase of the cell cycle and targets EZH2 in various cancers [75, 76], including HCCs [77, 78]. However, the underlying mechanisms of the regulation of EZH2 via miR-217 and how it affects malignant transformation still require further characterization.

Notably, recent studies have shown that the levels of circRNAs were increased in exosomes secreted from arsenite-transformed cells. The overexpression of miR-217 in normal cells reduced the expression of EZH2 (a promising biomarker of proliferation) [79, 80] and clclin-D1, which regulates the G1 to S phase transition in the cell cycle [81] (Fig. 2). This evidence suggests that exosomal circRNAs induced an accelerated cell cycle and promoted the proliferation of normal cells. Additionally, exosomal circRNAs secreted from adipose tissues can regulate the deubiquitination in HCC. Moreover, studies in vivo proved that overexpressed circ-deubiquitination (circ-DB) significantly downregulated miR-34a, leading to the activation of miR-34a/USP7/CyclinA2 signaling pathway. Consequently, exosomal circRNAs promote cell growth and suppress damage to DNA [63].

Intriguingly, a recent study demonstrated that exosomal circRASSF2 promoted laryngeal squamous cell carcinoma (LSCC) progression. Researchers have found circRASSF2 expression was significantly and consistently increased in LSCC tumor tissues compared with control groups [82]. Moreover, they knockdown exosomal circ-RASSF2 remarkably inhibited cell proliferation and migration via miR-302b-3p/IGF-1R axis, indicating the significance of exosomal circRNAs in tumor cell proliferation.

Overwhelming studies conformed that tumor onset and metastasis are associated with many oncogenes and that many oncogenic pathways are involved [83]. Nowadays, growing evidences indicate that cancer cell-to-cell communication and surrounding stroma contribute to metastasis. For example, Li et al. first identified that an important role of exosomal circ-PED8A in pancreatic ductal adenocarcinoma (PDAC). They found that a high expression of exosomal circ-PED8A in PDAC tissue was associated with lymphatic invasion, TNM stage and a poor survival rate. Further research revealed that circ-PDE8A promoted tumore cells growth via upregulating MET, which is a tyrosine kinase receptor, one of the key oncogenes for a subset of epithelial tumors, including PDAC [84]. Moreover, tumor-excreted circ-PDE8A could be released into blood circulation by exosome transportation, acting as a ceRNA for miR-338 to regulate MACC1 and promote invasive metastasis through the MACC/MET/ERK or AKT pathways (Fig. 3). Similarly, a recent study confirmed that circNRIP1 could be transmitted by exosome connection between gastric cancer (GC) cells, and exosomal circNRIP1 sponges miR-149-5p to affect the AKT1/mTOR signaling pathway and to promote the proliferation, migration and metastasis in gastric cancer [85]. Similarly, this phenomenon was also identified in breast cancer cells and patients [86]. These novel findings indicated the significance role of exosomal circRNAs in tumor metastasis.

Apart from the mentioned above, emerging evidence shows that the expression of certain exosomal circRNAs play important roles in the progression and metastasis of Hepatocellular carcinoma (HCC). For instance, Wang recently proved that exosomal circPTGR1 contributes to hepatocellular carcinoma metastasis [87]. Researchers found the high expression of exosomal circPTGR1 was related to the clinical stage and prognosis. In further, knockdown of exosomal circPTGR1 expression significantly suppressed tumor invasion and migration in non- and low-metastatic cell lines. According to bioinformatics analysis and both in vivo and in vitro experiments, exosomal circPTGR1 competed with MET interactions to target miR449a, leading to the dysregulation of tumor microenvironment and the promotion of HCC development.

Pathological epithelial mesenchymal transition (EMT) plays essential roles in tumor progression and the initiation of metastasis. Recently, Jie found that circ-IARS, secreted by pancreatic cancer cells and located in exosomes, was extensively expressed in pancreatic cancer tissues, and its expression level was responsible for tumor vasculature, tumor node metastasis (TNM) stage and liver metastasis [31]. Cancer cells must cross the endothelial barrier during extravasation for tumor distant metastasis [88]. Interestingly, the Ras homolog gene family, member A (RhoA), is associated with cytoskeleton regulation and reduces the expression of the junction ligand protein Zonula occludens-1 (ZO-1), leading to endothelial barrier dysfunction and endothelial solute permeability [89‐91]. It was indicated that the tumor suppressor miR-122 incudes mesenchymal-epithelial transition and inhibits the migration and metastasis of HCC by targeting RhoA [92]. Moreover, increased activity and expression of RhoA in human microvascular vein endothelial cells (HUVECs) was recently indicated to increase F-actin expression and decrease ZO-1 [93, 94], which damages the endothelial barrier function [89‐91] and reinforces endothelial permeability [95‐99]. In addition, Chen X et al. revealed that circPRMT5 was up-regulated in serum and urine exosomes from urothelial carcinoma of the bladder (UCB) tissues [100]. Further study confirmed exosomal circPRMT5 promoted UCB cell EMT via blocking with miR-30c, leading to the high expression of SNAIL1 and E-cadherin. Consequently, circRNAs can be transferred through exosomes from cancer cells to other cells as messengers, mediating cell-to-cell communication, regulating endothelial permeability, and thus promoting tumor metastasis.

Drug resistance is considered an enormous impediment in the treatment of cancer patients. As various underlying mechanisms are related to the progression of drug resistance, exosomes have attracted and gained universal attention as a novel therapeutic avenue [101, 102]. Importantly, exosomes transmit multi-drug resistance (MDR)-associated proteins [103] and noncoding RNAs to target cells [104], including circRNAs. For instance, Li L. et al. currently revealed serum exosomal circRNA (FECR1) was aberrantly upregulated in small cell lung cancer (SCLC) compared with control groups. In addition, serum exosomal- FECR1 was asscociated with clinical chemotherapy and poor survival in SCLC patients [105]. These evidences suggested a possible relationship between exosomal circRNAs dysregulation and drug resistance.

Accumulating evidence has shown that epithelial cancers, such as colorectal cancer (CRC), are preferentially resistant to many advanced drug therapies. Epidermal growth factor receptor (EGFR), a member of the HER-ErbB family, plays a significant role in the proliferation, angiogenesis, gene mutation and drug resistance in CRC. KRAS is an intracellular signaling molecule, and its mutations are found in approximately 40% of colorectal tumors [106]. Therefore, the identification of the key signaling pathways impacted by the KRAS mutation is beneficial for our understanding of how to pharmacologically target therapies.

Interestingly, the abundance of circRTN4 was remarkably upregulated in the exosomes derived from DLD-1 CRC cells, which possess a KRAS mutation compared with DKs-8 cells (wild-type KRAS). However, the intracellular abundance of circRTN4 was significantly downregulated in DLD-1 and DKO-1 cells, indicating exosomal circRNAs are independent of intracellular circRNA and may be actively transmitted between exosomes and the cytoplasm [60, 107]. These findings provide new insight into the function of exosomal circRNAs and demonstrate their ability to regulate chemotherapy resistance.

The nascent era of the clinical application of exosomel circRNA is growing rapidly. Emerging evidences suggest that exosomal circRNAs are released from various cells and carry signaling molecules for cellular communication, even the regulation of organ function. In addition, the expression profiles of exosomal circRNAs in patients distinguish from healthy groups, it is possible that exosomal circRNAs can function as molecular markers of some tumors to support diagnosis. Exosomal circRNAs are also being recognized as crucial mediators of healthy and diseased states and may become promising biomarkers of specific conditions. For instance, the levels of exosomal circRNAs were significantly up-regulated in DKs-8 cells compared to DLD-1 and DKO-1cells [60], suggesting that exosomal circRNAs may serve as a promising biomarker in colon cancers. Similarly, exosomal circular RNA IARS expression was higher than those of control groups both in pancreatic cancer tissues and in plasma exosomes [31], the result of this study indicated that the existence of exosomal circRNA could be a useful marker of PDAC diagnosis and prognostic prediction.

In addition, exosome-derived miRNA and proteomic materials may be used as diagnostic indicators of various tumors, such as ovarian, prostate and lung cancer [108‐110]. Unlike miRNA, circRNAs are exceptionally stable, conserved and have cell−/tissue-specific expression patterns, which suggest their potential applications as gene regulators as well as their possibility as molecular diagnostic and prognostic biomarkers. Importantly, the unique cellular stability and function of circRNAs to sponge miRNA and proteins may also indicate that circRNA is a promising vehicle for targeted drug delivery [111]. Despite its large potential, our understanding of exosomal circRNA is still in infancy.

Intriguingly, according to Li, 1215 circRNAs were identified in the human serum exosomes, and more than 90% of the detected circRNAs consisted of exons showed high stability and conserved exonuclease cleavage, suggesting that circRNAs can be actively transferred from cells to exosomes and indicating the underlying possibility of tumor diagnostic markers. Due to their applicability, specificity and accessibility, exosomal circRNAs have the potential for cellular therapy and for the theoretical concerns of neoplastic transformation from peripheric blood-, tissue-, or bone marrow derived stem cell transplantation.