Exosomes are 40-nm to 100-nm membrane vesicles that are secreted by various cells, and they play a major role in cell-cell communication. The objective of this study was to clarify the significance of the levels of microRNA in exosomes extracted from the sera of patients with esophageal squamous cell cancer (ESCC).
The authors isolated exosomes in serum samples from patients who had ESCC and from patients who had benign diseases without systemic inflammation. Total RNA was purified from the exosomes, and expression levels of microRNA-21 (miR-21) were analyzed by quantitative real-time polymerase chain reaction.
Serum exosomes from patients with ESCC induced the proliferation of ESCC cells in vitro. The expression levels of exosomal miR-21 were significantly higher in patients with ESCC than those with benign diseases with and without (C-reactive protein <0.3 mg/dL) systemic inflammation. MiR-21 was not detected in serum that remained after exosome extraction. Exosomal miR-21 expression was correlated with advanced tumor classification, positive lymph node status, and the presence of metastasis with inflammation or and clinical stage without inflammation (C-reactive protein <0.3 mg/dL).
Esophageal cancer with distant metastasis and local invasion is associated with a poor prognosis. Esophageal squamous cell carcinoma (ESCC) is a common pathologic cancer subtype in non-Western countries, and it can be caused by smoking and alcohol use. Combined treatment that includes surgery, chemotherapy, and radiation may improve clinical outcomes but is not curative. Early detection through frequent examinations, including endoscopic and radiologic examination, is the key to improved prognosis. Less complicated diagnostic methods, such as blood and urine ESCC screening, are not currently available.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate the translation of mRNAs to proteins.1-3 Several studies have revealed that miRNAs play an important role in multiple aspects of carcinogenesis through their oncogenic or tumor suppressor functions.4, 5 The identification of circulating miRNAs in the serum and plasma indicated that miRNAs may be potentially useful as clinical diagnostic or prognostic tools.6-8
Exosomes are small membrane vesicles secreted by most cell types, including T cells, platelets, and cancer cells.9-13 Exosomes contain proteins and nucleotides, and they are detected in a variety of body fluids and in in vitro cell culture medium.14, 15 Exosomes may act as a delivery system for cells, tissues, and organs, and they may regulate various bioactivities related to intercellular communication.16, 17 It remains unknown whether exosomes contain significant amounts of functional miRNAs in human samples.
We previously examined the expression of miRNA-21 (miR-21) in normal and tumor tissues from patients with ESCC using quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization analysis. These results indicated that miR-21 targets programmed cell death protein 4 (PDCD4) at the post-transcriptional level and regulates cell proliferation and invasion in ESCC.18 Next, we compared serum miR-21 expression levels in samples from patients with ESCC and from healthy controls using an miRNA purification system.19 The expression of serum miR-21 was detected at higher levels in patients with ESCC, indicating that miR-21 may be a biomarker for initial ESCC diagnosis. We hypothesized that exosomes in serum from patients with ESCC would contain large quantities of miRNA-21. To test this hypothesis, we purified exosomes from patient sera and examined their characteristics by transmission electron microscopy (TEM) and Western blot analysis. We also provided the first evidence of miR-21 enrichment in exosomes in serum from patients with ESCC and demonstrated that exosomal miR-21 in the serum was correlated with younger age, advanced tumor (T)-classification, positive lymph node status, and metastasis. Our data indicate that exosomal miR-21 may participate in ESCC progression, suggesting that it may be a useful therapeutic target or diagnostic biomarker of ESCC.
MATERIALS AND METHODS
Fifty-one patients with newly diagnosed ESCC (no prior treatment) at Kumamoto University Hospital from April 2009 to December 2010 were enrolled in the ESCC group in this study. Forty-one patients with benign diseases, including asymptomatic cholecystolithiasis and hernia, were included in the control group. Patients with other cancers were excluded from this study. All patients with ESCC underwent an upper gastroenterologic fiberscope examination, esophagography, enhanced computed tomography imaging from the neck to the abdominal region, and 18F-fluorodeoxyglucose-positron emission tomography studies for tumor staging according to TNM criteria. Blood examination and sampling were performed before treatment, which included surgery, chemotherapy, and radiotherapy. Informed consent was obtained from all participants for the use of their blood samples in this study. The study was approved by the Medical Ethics Committee of Kumamoto University.
Serum Samples and Reagents
Peripheral blood from patients was collected into ethylene diamine tetraacetic acid and centrifuged at 5000 revolutions per minute for 10 minuets at 4°C. The supernatants were transferred to fresh tubes and stored at −80°C before analysis. The primers for miRNA detection by PCR were purchased from Applied Biosystems (Foster City, Calif). Rabbit polyclonal antihuman cluster of differentiation 63 (CD63) immunoglobulin G (IgG) was purchased from Abcam (Tokyo, Japan).
Isolation of Exosomes From Serum
Exosomes were extracted from serum using ExoQuick Exosome Precipitation Solution (System Biosciences, Mountain View, Calif). Serum was obtained by centrifugation at ×3000g for 15 minutes to remove cells and cellular fragments, and subsequent filtration of the supernatant was accomplished through a 0.45-μm pore polyvinylidene fluoride filter (Millipore, Billerica, Mass). ExoQuick was added to the supernatants, and exosomes were precipitated by refrigeration at −20°C for 12 hours. Exosome pellets collected by centrifugation at ×1500g for 30 minutes were dissolved in 20 μL phosphate-buffered saline (PBS). Exosomes were quantified by using the micro BCA protein assay (Thermo Fisher Scientific KK, Tokyo, Japan).
Transmission Electron Microscopy
The samples were dissolved in HEPES (4-[2-hydroxyethyl]-1-piperazine ethanesulfonic acid) buffer, and a drop of the suspension was placed on a sheet of parafilm. A carbon-coated copper grid was floated on the drop for 10 seconds. Then, the grid was removed, and excess liquid was drained by touching the edge of the grid against a piece of clean filter paper. The grid was touched onto a drop of 2% uranyl acetate or phosphotungstic acid, pH 7.0, for approximately 5 seconds, and excess liquid was drained off. The grid was allowed to dry for several minutes and then examined using a JEM-1200 EX microscope (JEOL, Akishima, Japan) at 80 kiloelectron volts.
Western Blot Analysis
Extracted exosomes were washed once in PBS and lysed in a buffer containing 25 mmol/L Tris-HCl, pH 7.4; 100 mmol/L NaCl; 2 mmol/L ethylene diamine tetracetic acid; and 1% Triton-X 100 supplemented with 10 mg/mL aprotinin; 10 mg/mL leupeptin; 1 mmol/L Na3VO4; and 1 mmol/L phenylmethylsulfonylfluoride. The lysates were centrifuged, and the supernatants were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses were performed according to standard procedures. The membranes were incubated with polyclonal antihuman CD64 IgG followed by goat antirabbit IgG coupled to horseradish peroxidase (GE Healthcare, Madison, Wis). Peroxidase activity was observed using an enhanced chemiluminescence detection system (GE Healthcare).
MicroRNA Isolation From Exosomes
MiRNAs were isolated from exosomes using the mirVana microRNA isolation kit (Life Technologies, Carlsbad, Calif) and were eluted into 100 μL of heated elution solution according to the manufacturer's protocol. The purity and concentration of all RNA samples were quantified spectrophotometrically using the NanoDrop ND-1000 system (NanoDrop, Wilmington, Del), and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif).
MicroRNA Detection by Quantitative Real-Time Polymerase Chain Reaction
MiR-21 expression was determined by qRT-PCR using TaqMan microRNA assay kits (Applied Biosystems, Foster City, Calif). For combinational DNA (cDNA) synthesis, 10 ng total RNA from each serum sample were mixed with 0.15 mL of 100 mM deoxyribonucleotide triphosphate, MultiScribe reverse transcriptase (50 U/mL), 1.5 mL of 10X reverse transcription buffer, 0.19 mL RNase inhibitor (20 U/mL), and 1 mL gene-specific TaqMan primers. The reaction mixture was incubated at 16°C for 30 minutes, 42°C for 60 minutes, and 85°C for 5 minutes, as described previously.18 TaqMan qRT-PCR was performed in triplicate using the Light Cycler 480 System II (Roche Diagnostics, Basel, Switzerland) with a gene-specific primer/probe mix and DNA template. The reaction mixture was incubated at 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 1 second. The data were normalized to miR-16, an endogenous control gene that is stably expressed across samples. To evaluate the reproducibility of each experiment, the expression of miR-16 was measured using a synthetic nonhuman miRNA (Caenorhabditis elegans miR-39 miRNA [cel-miR-39]; Takara Bio Inc., Tokyo, Japan), which was spiked in to normalize the serum volume at the onset of RNA isolation, as described previously.20
Cell Proliferation Assay
ESCC cells (TE-1) were washed with PBS and suspended at 1 × 104 cells/mL in RPMI-1640 containing 1% fetal bovine serum. The cells were transferred into triplicate 96-well microtiter plates with or without exosomes isolated in sera from patients with ESCC. The plates were incubated for the indicated periods (see Results, below). The number of viable cells was assayed using the cholecystokinin-8 (CCK-8) test (Dojindo Molecular Technologies, Inc., Gaithersburg, Md) according to the manufacturer's protocol. Ten microliters of the CCK-8 solution, 2-(2 methoxy-4 nitro phenyl)-3-(4 nitro phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, were added to 100 μL of cells in the 96-well plates. Cell viability was determined by reading the optical density at 450 nm.
Differences in cell proliferation were analyzed with the Student t test. The expression of miR-21 in serum samples was compared using the Mann-Whitney U test or the Kruskal-Wallis test coupled with the Pearson correlation test, and the level of significance was set at P < .05. All statistical analyses were performed using the SPSS statistical software package (version 13.0; SPSS Inc., Chicago, Ill).
Characterization of Isolated Serum Exosomes by Transmission Electron Microscopy and Western Blot Analysis
To ensure the efficacy of the serum exosome isolation method, we characterized the microvesicles by TEM and Western blot analysis. Serum samples from patients with ESCC were obtained before and after treatment and observed with the ExoQuick exosome purification kit under TEM. Microvesicle clusters in the serum exhibited round vesicular membranes that measured 100 nm in greatest dimension (Fig. 1a,b). Microvesicle enrichment was observed in serum that had been treated with the ExoQuick kit (Fig. 1b). The expression of CD63, a tetraspanin family member that localizes to exosomal internal vesicles, was specifically observed as a dual band in isolated exosomes, and its intensity increased in a dose-dependent manner (Fig. 1c, lanes 1-4).
Confirmation of Exosomal MicroRNAs Using a Bioanalyzer
Exosomes contain a variety of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and miRNAs. The establishment of a strategy for purifying exosomal miRNAs from serum was essential for the accurate analysis of miRNA expression in this study. After serum exosome isolation, exosomal miRNAs were purified using the mirVana miRNA isolation kit. RNAs were separated by size and observed using a Bioanalyzer. Twenty-five-nucleotide-long small RNAs, representing miRNAs, were abundant. The 18S and 28S ribosomal RNAs were barely detectable (Fig. 1 day). These results suggest that our experimental method was specific for miRNAs. The exosomes isolated from serum contained miRNAs and were suitable and valuable for clinical sample analysis.
Serum Exosomes From Patients With Esophageal Squamous Cell Carcinoma Promote the Proliferation of TE-1 Cells
We investigated whether serum exosomes isolated from patients with ESCC could induce the proliferation of ESCC cell lines in vitro. Serum exosomes from Patient A with ESCC significantly increased the proliferation of TE-1 cells after incubation for 48 hours compared with untreated cells. Serum exosomes from Patient B with ESCC also significantly increased the proliferation of TE-1 cells after both 24 hours and 48 hours (Fig. 2). Serum exosomes from Patient A with ESCC significantly increased the proliferation of TE-1 cells for 48 hours compared with serum exosomes from patients with benign disease in TE-1 cells (data not shown). Serum exosomes from patients with ESCC were biologically active in TE-1 cells in vitro.
In general, exosomes contain a variety of proteins and RNAs. To identify miRNAs with potential for use as biomarkers in the initial diagnosis or prognosis of ESCC, we examined miRNA expression in serum exosomes from patients with ESCC.
Exosomal MicroRNA-21 Expression in Patients With Esophageal Squamous Cell Carcinoma
Initially, serum samples were collected from 51 patients with ESCC and from 41 patients with benign diseases, including cholecystolithiasis and inguinal hernia (ie, the control group). Age, sex, and C-reactive protein (CRP) levels differed significantly between the ESCC group and the control group (P = .0003) (Table 1). Exosomal miRNAs were obtained using a combination of the ExoQuick kit and the mirVana microRNA isolation system. Quantitative RT-PCR for miR-21 was performed, and the obtained values were normalized to miR-16 as an internal control. The expression of serum miR-21 in the ESCC group was significantly increased compared with its expression in the control group (P = .0009) (Fig. 3a,b). The expression of miR-21 was higher in exosomes and in the supernatant obtained by extraction of exosomes from both groups. The level of exosomal miR-21 was significantly higher than the miR-21 level in the supernatant among the patients with ESCC (data not shown).
Table 1. Characteristics of Patients With Esophageal Squamous Cell Carcinoma and Patients With Benign Diseases
Correlation Between Exosomal MicroRNA-21 Expression and the Clinicopathologic Features of Patients With Esophageal Squamous Cell Carcinoma
The 51 patients with ESCC were divided into high and low exosomal miR-21 expression groups (26 and 25 patients, respectively) using the median miR-21 value as the cutoff point. The median patient age (±standard deviation) was 68.0 ± 7.465 years. Seventy-five percent of patients were men in the high miR-21 expression group (high group), and 92.6% were men in the low miR-21 expression group (low group). The percentage of patients aged ≥68 years was higher in the high group than in the low group (64.7% vs percent; P = .0092). The percentage of patients with T3/T4 disease was higher in the high group than in the low group (80.8% vs 56%; P = .0422), the percentage of patients with positive lymph nodes was 80.8% in the high group and 68% in the low group, and the percentage of patients with metastasis was higher in the high group than in the low group (30.8% vs 12%; P = .0410). The percentage of patients with stage III/IV disease was 75% in the high group and 60.9% in the low group (Table 2). These results demonstrated that younger age, advanced T-classification, positive lymph node status, and metastasis were associated with higher exosomal miR-21 expression in serum from patients with ESCC.
Table 2. Characteristics of Patients With Esophageal Squamous Cell Carcinoma According to Exosomal MicroRNA-21 Expression
Exosomal MicroRNA-21 Expression in Patients With Esophageal Squamous Cell Carcinoma Without Systemic Inflammation
Recent publications have discussed the relevance of miR-21 in inflammation. We excluded patients with systemic inflammation (ie, those with CRP levels >0.3 mg/dL) to discriminate between miR-21 expression caused by cancer and that caused by inflammation. Exosomal miR-21 expression was examined by qRT-PCR in 30 patients with ESCC and in 30 patients with benign diseases. There were no significant differences regarding CRP levels (P = .0866) between the ESCC group and the control group except for age and sex (Table 3). The expression of exosomal miR-21 was increased significantly in the ESCC group compared with the control group (P = .01) (Fig. 4a,b).
Table 3. Characteristics of Patients With Esophageal Squamous Cell Carcinoma and Benign Diseases Without Inflammation (C-Reactive Protein Levels <0.3 mg/dL)
Correlation Between Exosomal MicroRNA-21 Expression and Clinicopathologic Factors in Patients With Esophageal Squamous Cell Carcinoma Without Systemic Inflammation
The 30 patients with ESCC were divided into high and low exosomal miR-21 expression groups with 15 patients in each group. The median patient age (±standard deviation) was 68.4 ± 8.029 years (Table 3), 55% of patients were men in the high miR-21 expression group (high group), and 86.7% were men in the low miR-21 expression group (low group).
The percentage of patients aged ≥68 years was higher in the high group than in the low group (77.6% vs 60%; P = .0092). The percentage of patients with T3/T4 disease was 80% in the high group and 65.3% in the low group, the percentage of patients with positive lymph nodes was 77.6% in the high group and 77.6% in the low group, and the percentage of patients with metastasis was lower in the high group than in the low group (66.7% vs 86.7%; P = .0410). The percentage of patients with stage III/IV disease was 66.7% in the high group and 53.3% in the low group (Table 4). These results demonstrated that older age, negative metastasis, and clinical disease stage were associated with higher exosomal miR-21 expression in the serum from patients with ESCC.
Table 4. Characteristics of Patients With Esophageal Squamous Cell Carcinoma Without Inflammation (C-Reactive Protein Levels <0.3 mg/dL) According to Exosomal MicroRNA-21 Expression
Exosomes were originally described in 1983, and recent studies have indicated that exosomes contain small RNAs, particularly miRNAs.21-23 Exosomal miRNAs with various biologic functions are secreted by origin cells to silence target mRNAs in other target cells.15 Thus, exosomal miRNAs are involved in intercellular communication to regulate gene expression.24
Several methods for the detection and purification of exosomal or circulating miRNAs have been reported.21, 25-28 However, the purity and identity of the materials used in those studies were not well validated. In the current study, we initially confirmed that our miRNA and exosome isolation system produced material that was enriched for exosomal miRNAs. The characteristic size and shape of the exosomes were observed by TEM (Fig. 1a,b), and Western blot analysis demonstrated that the exosomes expressed the exosome-specific CD63 protein (Fig. 1c). The high quality of the purified exosomal miRNAs was confirmed using a Bioanalyzer (Fig. 1d). To our knowledge, this is the first report to demonstrate that serum exosomes isolated from patients with ESCC are biologically active in ESCC cells in vitro. Exosomes are involved in the regulation of intracellular signal transduction and gene expression through cell-cell communication. The molecular components of exosomes may affect cancer progression by modulating carcinogenesis, growth, invasion, and metastasis. Exosomes derived from gastric cancer cells reportedly promote cell proliferation through phosphatidylinositol 3-kinase/v-Akt murine thymoma viral oncogene homolog (PI3/Akt) and mitogen-activated protein kinase-Erk kinase/extracellular signal-regulated kinase (MAPK/ERK) activation.34 We have not yet confirmed whether exosomal miR-21 induces cell proliferation, for example, by antisense or inhibitor approaches. Our previous study demonstrated that miR-21 suppressed PDCD4 mRNA expression and promoted cell proliferation in vitro.18 In the current study, we hypothesized that exosomal miR-21 effectively is transferred to cancer cells and that miR-21 promotes cancer progression as an oncomir.
Our previous results demonstrated that circulating miR-21 was enhanced in sera from patients with ESCC compared with healthy control donors. However, a re-evaluation of the detection of circulating miR-21 in serum by other approaches was necessary for 2 reasons. First, the donors in our healthy control group were younger than the patients in our ESCC group. Second, and most important, the experimental methods may not have detected miRNA in the exosomes alone; the detected miR-21 may have originated from the exosomes of viable cells or the cytoplasm of damaged cells. To discriminate between exosomal miRNAs and nonexosomal circulating miRNAs, we purified exosomal miRNAs from serum after the isolation of miRNAs. We observed that miR-21 expression was significantly higher in exosomes than in the serum remaining after exosome extraction, indicating that the circulating miR-21 originated from exosomes. Previous reports indicated that some miRNAs were detected in serum as well as in exosomes and that the exosomes in serum are highly enriched in miRNAs.35
Figure 3 demonstrates that exosomal miR-21 levels in the ESCC group were significantly increased compared with levels in the control group. This relation was consistent with previous data regarding circulating miRNAs. Patients with ESCC and those with benign diseases may suffer from systemic inflammation. MiR-21 expression may be involved in acute and chronic inflammatory diseases, such as infection, rheumatoid arthritis, and inflammatory bowel disease. To avoid the complicating effects of miR-21 induction by inflammation, we excluded patients who had CRP levels >0.3 mg/dL. Figure 4 demonstrates that exosomal miR-21 expression in the ESCC group was up-regulated compared with expression in the control group with no inflammation.
Exosomal miR-21 expression is correlated with clinicopathologic data in Tables 1 through 4. Our previous report18, 19 regarding miR-21 from ESCC tissues indicated that miR-21 functions as an oncogenic miRNA that contributes to the progression of ESCC (eg cell proliferation and invasion) by suppressing target genes like PDCD-4, which binds to and inhibits the translation initiation factor eukaryotic initiation factor 4a, thereby impacting protein translation. MiR-21 expression is not specific to ESCC, and its overexpression has been reported in a variety of human cancer cells, including colon,29 liver,30 pancreas,31 ovarian,32 and prostate cancer cells.33 MiR-21 appears to be a key player in carcinogenesis, similar to the p53 gene. We previously described that the effectiveness of chemotherapies, such as docetaxel, cisplatin, and 5-fluorouracil, as well as surgical procedures were connected to the reduction in serum miR-21 expression.19 Serum miR-21 levels were higher in patients who were resistant to docetaxel-based chemotherapy compared with patients who were sensitive to chemotherapy.36 The effectiveness of various anticancer drugs may be associated with reduced serum miR-21 expression.
Therefore, we conclude that exosomal miR-21 may be a useful biomarker for detecting ESCC progression or for evaluating the effectiveness of ESCC treatment. Exosomes from patients with ESCC may participate in disease progression by allowing ESCC to escape immune surveillance. Constitutive exosomal miR-21 overexpression may be associated with poor survival in patients with ESCC.
In summary, we observed up-regulated expression of exosomal miR-21 in serum from patients with ESCC compared with its expression in serum from a benign control group in which inflammation was absent. Exosomal miR-21 expression is related to clinicopathologic findings, indicating that an analysis of the regulation of exosomal miR-21 may be relevant for the treatment of ESCC. Moreover, exosomal miRNAs, particularly miR-21, are useful targets for cancer therapy.
We thank Masaya Saitou, Hirohisa Okabe, Yoshifumi Baba, and Masaaki Iwatsuki for their invaluable comments. We are grateful to the Hanaichi UltraStracture Research Institute for technical support with transmission electron microscopy.
This work was supported in part by the following grants and foundations: Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (grant 23791550); Takeda Science Foundation, 2010; Okukubo Memorial Fund for Medical Research in Kumamoto University School of Medicine, 2010; Uehara Memorial Foundation, 2010; and Yokoyama Foundation for Clinical Pharmacology, 2011.