Exosomal mRNAs are important regulators of cellular biological processes. Exosomal mRNAs were first identified in mouse MC/9 and human HMC-1 cell lines using microarray analysis. Interestingly, exosomal mRNAs from mouse mast cells could be transferred into human mast cell lines, indicating that exosomes are effective vessels for the delivery of mRNA to other cells. It was further discovered that mRNAs were selectively taken up into exosomes because 270 transcripts were only detected in exosomes other than the donor cells (MC/9). Additionally, exosomal mRNAs were translated into functional proteins in the recipient cells, suggesting that exosomal mRNAs retain their function in recipient cells. These results demonstrate that exosomal mRNAs are critical mediators of intercellular communications [2].

Exosomal mRNAs also have advantages as biomarkers. First, exosomal mRNA can reflect the conditions of donor cells and are easy to detect since some exosomes (e.g., blood exosomes) circulate throughout the entire body [39]. In addition, the membrane of the exosome can protect exosomal mRNA from digestion by RNases [40]. For example, urinary exosomal mRNAs can remain stable for as long as two weeks at 4 °C [41]. Finally, exosomal mRNAs can affect the function of recipient cells more directly than exosomal ncRNAs since they can be translated into proteins in recipient cells.

Exosomal mRNAs are considered as a critical indicator of cancers. Previous studies reported that tumor cells can express tumor-specific mRNAs or change the expression levels of normal exosomal mRNAs. For example, in glioblastoma, epidermal growth factor receptor (EGFR) is expressed by the tumor-specific mRNA EGFRvIII, which was recommended as a diagnostic biomarker for glioblastoma [42]. As another example, telomerase is considered a hallmark of cancer [43]. Human telomerase reverse transcriptase (hTERT) is generally not expressed in healthy humans. However, hTERT is detectable in multiple cancers, such as acute myelocytic leukemia, Burkitt lymphoma, and chronic lymphocytic leukemia, indicating that serum exosomal hTERT mRNA may be a potential pan-cancer biomarker [44]. Additionally, serum exosomal heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) mRNA levels in hepatocellular carcinoma (HCC) patients were significantly higher than those in control groups. Thus, exosomal hnRNPH1 was suggested as a potential biomarker for HCC diagnosis [45].

Exosomal mRNAs were also suggested as biomarkers for diagnosing other diseases, such as those related to the human central nervous system and urinary system. A study including 20 older healthy adult subjects (≥65 years) and 20 younger healthy adult subjects (21–45 years) indicated that amyloid-β1-42 peptide (Aβ)–the main component of the amyloid plaques found in the brains of patients with Alzheimer’s disease [46] stimulated the release of exosomal cytokine mRNAs via macrophages and CD4 memory T-cells, indicating that exosomal cytokine mRNAs could potentially act as diagnostic biomarkers for Alzheimer’s disease [47]. Furthermore, Lv et al. suggested the urinary exosomal mRNA CD2 associated protein (CD2AP) as a biomarker for the diagnosis of kidney disease since a decrease in its expression level reflects the severity of tubulointerstitial fibrosis and glomerulosclerosis [48].

Exosomal mRNAs can also be used as biomarkers for the evaluation of drug resistance., which is currently one of the major challenges in cancer therapy. Shao et al. analyzed the exosomal mRNAs in the serum of 32 individuals (17 glioblastoma multiforme patients and 15 healthy individuals) and found that the exosomal O-6-methylguanine-DNA methyltransferase (MGMT) and N-methylpurine DNA glycosylase (APNG) mRNA levels were correlated with the levels of temozolomide resistance and the treatment efficacy in glioblastoma multiforme patients [49].

Based on studies of intracellular mRNAs, two successful commercial kits use urinary exosomal mRNAs (SAM pointed domain-containing ETS transcription factor (SPDEF) and ETS transcription factor (ERG)) and plasma exosomal mRNA (echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion transcripts) to detect prostate cancer and nonsmall-cell lung cancer, respectively [50,51,52], which demonstrates the functionality of exosomal mRNAs as biomarkers.

Some examples of exosomal mRNAs with the potential to be used as biomarkers for disease diagnosis are summarized in Table 1.

MiRNAs, a class of small noncoding RNAs with a length of ~22 nt, play a principal role in the regulation of gene expression at the post-transcriptional level [61]. MiRNAs mainly function by binding to the 3′untranslated region (3′-UTR) of target mRNAs and inducing cleavage or reducing translation [62]. Both cellular miRNAs and exosomal miRNAs are involved in various biological activities, including cancer progression, immune responses, and cell cycle progression [61].

Exosomal miRNAs were proposed as potential biomarkers for diagnosing and predicting diseases. This primarily relies on their ability to reflect the internal conditions of cells, including physiological and pathological conditions [63]. In addition, miRNAs are the most abundant RNA molecules in exosomes, which makes their detection easier than that of other types of exosomal RNA [64]. Lastly, exosomal miRNAs show improved stability due to the protection afforded by their encapsulating membranes. It was observed that exosomal miRNAs can remain stable for five years when stored at −20 °C or for 14 days at 4 °C, with this stability being unaffected by repeated freezing and thawing [65]. These characteristics help increase the sensitivity of exosomal miRNA-based biomarkers. This is critical as limited sensitivity results in low detectability.

Recent studies identified some exosomal miRNAs with great potential for diagnosing cancers. Exosomes derived from cancer cells might affect the function of normal cells via miRNAs and could be an essential factor driving cancer metastasis. In a study on brain cancer, human and mouse tumor cells were observed to stop expressing phosphatase and tensin homolog (PTEN)—an important tumor suppressor—after dissemination to the brain due to inhibition mediated by exosomal miR-19 that was secreted by astrocytes, which indicates that exosomal miR-19 might be a suitable biomarker for diagnosing brain cancer metastasis [66]. Another study showed that colorectal cancer cells promoted the M2 pole of macrophages by transferring a set of miRNAs (miR-25-3p, miR-130b-3p, and miR-425-5p) through exosomes in response to stromal cell-derived factor 1/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) activation through the PTEN/PI3K/Akt pathway, which enhanced the liver metastasis of colorectal cancer in vitro and in vivo [67].

Exosomal miRNAs can serve as biomarkers for diagnosing diseases other than cancer. Macrophages are critical for the maintenance of metabolic homeostasis, and their exosomal miRNAs are closely related to various metabolic-related diseases, such as diabetes and obesity [68]. For instance, exosomal miR-690 binds to the 3′-UTR of the NAD kinase (NADK) mRNA, which is responsible for regulating insulin signaling and macrophage inflammation, to enhance insulin sensitivity [69]. Exosomal miRNAs could also reflect the viral infection of cells. One well-known example is the Epstein–Barr virus (EBV), the first human virus that was found to encode miRNAs [70]. B cells infected with EBV secrete exosomes containing EBV-miRNAs, which affect gene expression in the recipient cells [71].

Other recent examples of exosomal miRNAs with the potential to be used as biomarkers for disease diagnosis are summarized in Table 2.

Long noncoding RNAs (lncRNAs) are a type of noncoding RNA longer than 200 nt [99]. LncRNAs are involved in the regulation of gene expression in diverse manners at multiple levels, such as gene transcription control, chromatin structure modulation, RNA splicing regulation, miRNA sponging, and RNA-binding protein interaction [100].

Typically, exosomal lncRNAs display strong tissue specificity and poor conservation, and the expression levels of exosomal lncRNAs can indicate the health conditions affecting tissues and cells, making them suitable for use as biomarkers [101,102]. Additionally, the large number of tissue- and cell-specific lncRNAs provides many options for diagnostic biomarkers.

Exosomal lncRNAs were suggested as biomarkers to diagnose diseases. For example, lncRNA prostate cancer antigen 3 (lncPCA3)—found in urinary exosomes—was approved as a biomarker to diagnose human prostate cancer by the US Food and Drug Administration. Exosomal lncPCA3 shows a much higher expression level in prostate cancer cells than in inflamed or normal prostate tissue (up to 70- to 100-fold), making lncPCA3 an efficient biomarker for diagnosing prostate cancer [103,104,105]. Another example is H19, a well-known oncogenic lncRNA found in serum exosomes that was significantly upregulated in bladder cancer patients when compared to that of healthy individuals, which highlights its potential use as a biomarker for bladder cancer [106,107].

Compared to exosomal miRNA, the study of exosomal lncRNA is in its infancy. According to GENCODE, there are more than 16,000 lncRNA genes in the human genome, which are estimated to produce more than 10,000 lncRNA transcripts, indicating a considerable candidate pool of potential diagnostic biomarkers [108,109].

Recent examples of exosomal lncRNAs that are potential diagnostic biomarkers are summarized in Table 3.

Circular RNAs (circRNAs) are a subset of noncoding RNAs that lack 5′ caps and 3′ poly(A) tails and instead have a closed-loop structure [126]. As a class of endogenous RNAs, circRNAs are involved in various biological processes, including alternative splicing, transcription regulation, miRNA sponging, protein scaffolding, interacting with RNA-binding protein (RBP), and pseudogene creation [127,128,129,130,131]. Due to their multiple functions, circRNAs were closely linked to many diseases, such as cancers, neurodegeneration, diabetes, cerebrovascular diseases, and cardiovascular diseases. Thus, the expression of circRNAs could reflect the presence of these diseases [132,133,134,135]. Additionally, the closed-loop structure provides circRNAs with resistance to exoribonucleases and a long half-life [136,137,138]. These characteristics make exosomal circRNAs ideal biomarkers for disease diagnosis.

Exosomal circRNAs were studied as biomarkers for cancer diagnosis. RNA-seq analysis demonstrated that exosomal circRNAs enter circulation and are enriched at least two-fold in exosomes when compared to their levels in donor cells [139]. A study of exosomal circRNAs in colorectal cancer patients and healthy individuals revealed 67 absent circRNAs and 257 new circRNAs in patient serum exosomes, indicating the potential of exosomal circRNAs to act as biomarkers for the diagnosis of colorectal cancers [139]. Another study using microarray sequencing found a significant decrease in plasma exosomal circ-0051443 in patients with hepatocellular carcinoma (HCC), and it was shown that circ-00551443 releaseed BCL2 antagonist/killer 1 (BAK1), which initiated cell apoptosis to prevent HCC via sponging miR-331-3p [140]. As a result, circ-0051443 was considered a tumor suppressor and a novel potential biomarker for HCC diagnosis [140]. As another example, plasma exosomal circ-133 can be used as a biomarker to monitor colorectal tumor progression, as exosomal circ-133 expression induced by hypoxia was able to sponge miR-133a to activate the GEF-H1/RhoA axis in normoxic colorectal cancer cells, leading to the migration of colorectal cancer cells [141].

Exosomal circRNAs can also act as diagnostic biomarkers for nervous system diseases, ischemic diseases, and cardiovascular diseases. In cerebrospinal fluid, 26 exosomal circRNAs were shown to have significantly different expression levels in patients with immune-mediated demyelinating disease (IMDD) when compared to that of healthy controls, while the upregulations of hsa_circ_0087862 and hsa_circ_0012077 were recommended as potential diagnostic biomarkers for IMDD [138]. After ischemia, vascular smooth muscle cells secrete exosomal circRNA cZFP609, which is delivered into endothelial cells, resulting in reduced vascular endothelial growth factor A (VEGFA) expression and disrupted endothelial angiogenic function via the interaction with and sequestration of hypoxia-inducible factor 1 subunit alpha (HIF1α) [142]. In this case, cZFP609 may act as a suitable biomarker to assess the clinical outcome and prognosis of ischemic diseases [142].