Oesophageal carcinoma is the sixth leading cause of cancer-related deaths worldwide ( Jemal et al , 2011 ). Although there are two histological types of oesophageal carcinoma, oesophageal squamous cell carcinoma (ESCC) is the predominant histological type in Asian countries and accounts for approximately 90% of oesophageal carcinomas ( Hiyama et al , 2007 ) and is one of the most aggressive carcinomas of the gastrointestinal tract. Although surgical techniques, perioperative management, and perioperative chemo and/or radiotherapy regimens have greatly progressed, even now ESCC continues to present with an extremely poor prognosis. Therefore, primary tumours must be detected at an early stage and treated with curative intent to improve survival rates, and patients with far advanced disease must be diagnosed preoperatively in order to avoid impairments in their quality of life following unnecessary extended surgery. Moreover, recurrent disease must be diagnosed when it is still minimal or clinically occult to improve the prognosis of ESCC patients.
Because identifying molecular targets for ESCC treatment may contribute to the improvement of survival of patients with this lethal disease, several recent studies have clarified that certain molecules, such as COX-2 , BCL-2 , TP53 , p16 , cyclin D1 , FAS , EGFR , VEGF , and E-cadherin ( Hollstein et al , 1990 ; Adélaide et al , 1995 ; Gratas et al , 1998 ; Meng et al , 2011 ; Shi et al , 2011 ), have important roles in tumorigenesis and the development of ESCC. Moreover, various genetic and epigenetic alterations that contribute to the carcinogenesis of ESCC have been elucidated. In clinical settings, however, only a few molecules have been validated as diagnostic, therapeutic, and/or prognostic biomarkers for ESCC. Conventional serum tumour markers, such as carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC), have been used in convenient diagnostic assays ( Kosugi et al , 2004 ; Mroczko et al , 2008 ) for the early detection of ESCC and the monitoring of tumour dynamics. These serum tumour markers, however, lack sufficient sensitivity and specificity to enable the early detection of ESCC. Hence, development of novel molecular biomarkers using less invasive technology is necessary and could allow clinicians to detect early ESCC, monitor tumour dynamics, and predict treatment sensitivity and prognosis.
MicroRNAs (miRNAs), which are small non-coding RNAs, regulate the translation of specific protein-coding genes. Since their discovery in 1993 ( Lee et al , 1993 ), miRNAs have been intensively studied in cancer research, and these molecules are predicted to control as much as 30% of all gene expression ( Lewis et al , 2005 ). Thus altered miRNA expression has been associated with several diseases, and tumour miRNAs are involved in tumorigenesis and the development of various cancers ( He et al , 2005 ; Lu et al , 2005 ; Calin and Croce, 2006 ; He et al , 2007 ). Recently, several studies have identified that miRNAs are detectable in plasma/serum ( Calin and Croce, 2006 ; Chen et al , 2008 ; Filipowicz et al , 2008 ; Ichikawa et al , 2012 ). Tumour-derived miRNAs are resistant to endogenous ribonuclease activity, because these may bind to proteins, such as the Argonaute 2 protein and high-density lipoprotein ( Arroyo et al , 2011 ; Vickers et al , 2011 ), or may be packaged by secretory particles, including apoptotic bodies and exosomes in plasma/serum ( Hasselmann et al , 2001 ; Mitchell et al , 2008 ; Cocucci et al , 2009 ; Kosaka et al , 2010 ). Therefore, miRNAs can be present in a remarkably stable form ( Mitchell et al , 2008 ; Zhu and Fan, 2011 ), and the expression levels of serum miRNAs are reproducible and consistent among individuals ( Chen et al , 2008 ; Mitchell et al , 2008 ). Furthermore, secretory vesicles, which include specific miRNAs, can function as intercellular transmitters. Namely, secreted miRNAs from donor cells can be transferred to and function in recipient cells ( Valadi et al , 2007 ; Skog et al , 2008 ; Rechavi et al , 2009 ).
Regarding ESCC, several research groups, including our own, have reported the potential utility of circulating miRNAs in plasma/serum in clinical application ( Zhang et al , 2010 ; Komatsu et al , 2011 , 2012 ; Kurashige et al , 2012 ; Hirajima et al , 2013 ; Takeshita et al , 2013 ). Each identified miRNA in the plasma/serum of ESCC patients could be valuable for detecting cancer, monitoring tumour, and predicting prognosis. However, these reported blood-based miRNAs do not always indicate all candidates for ESCC, and more sensitive and promising candidate miRNAs for screening cancer, monitoring the tumour status, and predicting prognosis could be found in clinical settings. Therefore, we performed a genome-wide miRNA array-based approach to detect novel plasma miRNA candidates for detecting ESCC and monitoring the tumour status.
In this study, we selected eight upregulated and common plasma miRNA candidates (miR-15b, 16, 17, 25, 19b, 20a, 20b, and 106a) through the integration of two miRNA array-based approaches, which were used to compare plasma miRNA levels between ESCC patients and healthy volunteers and between preoperative and postoperative ESCC patients, respectively. We validated that plasma miR-25, which is located in the miR-106-25 cluster, is overexpressed in ESCC tissues ( Guo et al , 2008 ; Zhu et al , 2011 ; Xu et al , 2012 ; Fang et al , 2013 ), and has an oncogenic function to negatively regulate tumour suppressive genes such as PTEN , p21 , TP53 , Bim , CDH1 , and DR4 ( Petrocca et al , 2008 ; Kan et al , 2009 ; Li et al , 2009 ; Poliseno et al , 2010 ; Kumar et al , 2011 ; Razumilava et al , 2012 ; Xu et al , 2012 ), was upregulated in ESCC patients and preoperative ESCC patients. Moreover, we clearly demonstrated that plasma miR-25 levels are useful to detect ESCC and monitor tumour dynamics for tumour resection and recurrence. Our results provided evidence that plasma miR-25 levels contribute to clinical decision making in ESCC treatments to a clinically satisfactory degree.
Materials and methods
Patients and samples
This study was approved by the Institutional Review Board of Kyoto Prefectural University of Medicine, and each subject provided written informed consent. Between November 2008 and June 2011, 105 consecutive preoperative plasma samples were collected from consecutive ESCC patients who underwent curative oesophagectomy at the Kyoto Prefectural University of Medicine. Patient characteristics with respect to age, sex, venous invasion, lymphatic invasion, T stage, N stage, disease stage, and resection status are described in Table 1 . Moreover, additional 20 consecutive pretreatment plasma samples were collected from consecutive superficial ESCC patients who underwent curative endoscopic resection (m2-sm1, R0). All patients were pathologically diagnosed with ESCC using surgical specimens and biopsies. Ten ESCC specimens were collected from patients undergoing oesophagectomy, and seven normal oesophageal tissue specimens of the abdominal esophagus were collected from patients undergoing total gastrectomy for gastric cancer; these patients were selected for the normal specimens because the non-cancerous oesophageal tissues of ESCC patients may still exhibit dysplasia or potentially be cancerous tissue. As a control, plasma was collected from 50 healthy volunteers. These healthy volunteers included medical personnel and patients with benign diseases, such as cholelithiasis. These patients underwent medical examinations, including endoscopy, and were found not to have any oesophageal disease or other cancerous disease. Tumour stages were assessed according to the Union of International Control of Cancer (UICC) classification ( Sobin et al , 2009 ). The details of plasma samples were summarised in Supplementary Figure S1 .
Peripheral blood (7 ml) was obtained from each patient at the time before the oesophagectomy or endoscopic resection and from the healthy volunteers. Blood was collected from patients and controls in sodium heparin tubes (BD Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and immediately subjected to the three-spin protocol (1500 r.p.m. for 30 min, 3000 r.p.m. for 5 min, and 4500 r.p.m. for 5 min) to prevent contamination by cellular nucleic acids. The plasma samples were stored at −80 °C until further processing. The resected specimens were fixed in formalin and embedded in paraffin for pathological diagnosis. Macroscopic and microscopic classification of tumours was based on the UICC/TMN staging system ( Sobin et al , 2009 ).
RNA extraction
Total RNA was extracted from 400 μ l of plasma using the mirVana PARIS Kit (Ambion, Austin, TX, USA) and finally eluted into 100 μ l of preheated (95 °C) Elution Solution according to the manufacturer's protocol. Using the formalin-fixed paraffin-embedded tissues, total RNA was extracted from four 15- μ m-thick slices of tissue (total 60 μ m in thickness) using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion) and then eluted into 60 μ l of Elution Solution according to the manufacturer's protocol.
miRNA microarray analysis
Microarray analyses of the plasma samples were performed using the 3D-Gene miRNA microarray platform (Toray, Kamakura, Japan) ( Nagino et al , 2006 ; Giovannetti et al , 2012 ; Konishi et al , 2012 ). RNA extraction was performed according to the manufacturer's instructions described previously ( Konishi et al , 2012 ). Briefly, the amount of total RNA in plasma was too small, and so 2 of the 4 μ l of extracted total RNA from 300 μ l of plasma samples were used in the microarray experiments. The reason why the volume of 300 μ l plasma was used as the common denominator in each microarray analysis is that there was no definite internal control in plasma miRNA analyses as shown in our previous studies ( Tsujiura et al , 2010 ; Komatsu et al , 2011 ; Morimura et al , 2011 ; Komatsu et al , 2012 ; Konishi et al , 2012 ; Hirajima et al , 2013 ; Kawaguchi et al , 2013 ). This RNA was labelled with Hy5 using the Label IT miRNA labeling kit (Takara Bio, Otsu, Japan) and hybridised at 32 °C for 16 h on the 3D-Gene chip. The 3D-Gene miRNA microarray (Human_miRNA_17v1.0.0, Toray Industries, Kamakura, Japan) can mount >1500 miRNAs based on the Human miRNA Version17 of MirBase ( http://microrna.sanger.ac.uk/ ). Microarray was scanned, and the obtained images were numerated using the 3D-GeneH scanner 3000 (Toray Industries). The expression level of each miRNA was globally normalised using the background- subtracted signal intensity of the entire miRNAs in each microarray. The obtained microarray images were analysed using GenePix Pro TM (Molecular Device, Sunnyvale, CA, USA).
In these miRNA array-based analyses, two different approaches were performed. One approach was used to compare plasma miRNA levels derived from 300 μ l of the plasma sample between ESCC patients and healthy volunteers. Namely, each 100 μ l plasma sample from three ESCC patients was equally mixed, and totally 300 μ l of plasma sample was used as a sample of ESCC patients. On the other hand, each 60 μ l plasma sample from five healthy volunteers was equally mixed, and totally 300 μ l of plasma sample was used as a healthy volunteers' sample. The second approach was used to compare plasma miRNA levels between paired preoperative and postoperative ESCC patients. Each 100 μ l plasma sample from three preoperative or postoperative ESCC patients was equally mixed, respectively, and totally 300 μ l of a plasma sample was used as a sample of preoperative or postoperative ESCC patients.
Protocol for quantification of miRNA by quantitative RT–PCR (qRT–PCR)
Test-scale analyses were performed using qRT–PCR in order to validate the utility of selected candidates from miRNA array-based approaches. The amounts of miRNAs in the plasma were quantified by qRT–PCR using human TaqMan MicroRNA Assay Kits (Applied Biosystems, Foster City, CA, USA). The reverse transcription reaction was carried out using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) in 15 μ l solution containing 5 μ l of RNA extract, 0.15 μ l of 100 m M dNTPs, 1 μ l of MultiScribe reverse transcriptase (50 U μ l −1 ), 1.5 μ l of 10 × reverse transcription buffer, 0.19 μ l of RNase inhibitor (20 U μ l −1 ), 1 μ l of gene-specific primer (has-miR-25, Assay ID: 000403; has-miR-16, Assay ID: 000391; has-miR-20b, Assay ID: 001014; has-miR-106a, Assay ID: 002169; has-miR-17, Assay ID: 002308; has-miR-20a, Assay ID: 000580; has-miR-15b, Assay ID: 000390; has-miR-19b, Assay ID: 000396; and RNU6B, Assay ID: 001093), and 4.16 μ l of nuclease-free water. For cDNA synthesis, the reaction mixtures were incubated at 16 °C for 30 min, at 42 °C for 30 min, and at 85 °C for 5 min and then held at 4 °C. Next, 1.33 μ l of cDNA solution was amplified using 10 μ l of TaqMan 2 × Universal PCR Master Mix with no AmpErase UNG reagent (Applied Biosystems), 1 μ l of gene-specific primer/probe, and 7.67 μ l of nuclease-free water in a final volume of 20 μ l. Quantitative PCR was run on a 7300 Real-time PCR system (Applied Biosystems), and the reaction mixtures were incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Cycle threshold (Ct) values were calculated using the SDS 1.4 software (Applied Biosystems).
Plasma miRNA levels were calculated on a standard curve constructed using synthetic miRNAs from the mirVana miRNA Reference Panel (Ambion). Standard reference miRNAs were amplified by RT–PCR of a 10-fold serial dilution of the mirVana miRNA Reference Panel. The linearity of quantitative RT–PCR was confirmed between the logarithm of the amount of input miRNA and Ct values for a range of concentrations (1–0.0001 fmol) of each synthetic miRNA.
Although some investigators have determined plasma miRNAs levels by comparing with internal control miRNAs ( Ng et al , 2009 ), it remains controversial as to which miRNAs are suitable as internal controls for plasma assays. Therefore we confirmed a linear correlation between the logarithm of the amount of input synthetic miRNA and the Ct value using real-time PCR, as well as the feasibility of extracting total RNA and amplifying specific miRNA in plasma samples. On the basis of these findings, we utilised the absolute concentration for measuring plasma miRNA in this study.
As the expression of miRNAs from tissue samples was normalised using the 2−ΔΔCT method relative to U6 small nuclear RNA (RNU6B), ΔCt was calculated by subtracting Ct values of RNU6B from those of the miRNAs of interest. ΔΔCt was then calculated by subtracting ΔCt of normal tissue from ΔCt of ESCC tissues. The change in gene expression was calculated using the equation 2−ΔΔCt ( Livak and Schmittgen, 2001 ).
ESCC cell lines and culture
ESCC cell lines such as TE2, TE9, TE13, and KYSE70 and the fibroblast cell line WI-38 were purchased from RIKEN Cell Bank (Tsukuba, Japan). TE2, TE9, TE13, and KYSE70 cells were cultured in Roswell Park Memorial Institute-1640 medium (Sigma, St Louis, MO, USA). WI-38 cells were cultured in Dulbecco's Minimum Essential Medium. All media were purchased from Sigma and supplemented with 100 ml l −1 fetal bovine serum (Trace Scientific, Melbourne, Victoria, Australia). All cell lines were cultured in 50 ml l −1 carbon dioxide at 37 °C in a humidified chamber.
Statistical analysis
In miRNA array-based analyses, the signal intensity ratio and log2 ratio of each plasma miRNA were calculated by the ratio of ESCC patients/healthy volunteers or pretreatment ESCC patients/postoperative ESCC patents. The Mann–Whitney test was used to compare differences in plasma miRNA levels between the cancer and healthy groups, and the Wilcoxon's test was used to compare plasma levels between the paired samples before and 1 month after oesophagectomy or between the samples after oesophagectomy and at recurrences. A P -value of 0.05 was considered significant. The area under the receiver operating characteristic (ROC) curve (AUC) was used to assess the feasibility of using plasma miRNA levels as a diagnostic tool for detecting ESCC. The Youden index was used to determine the cutoff value for plasma miRNA levels ( Akobeng, 2007 ).
Results
Study design to detect novel plasma miRNA biomarkers for ESCC
This study was divided into several parts: (1) Selection of miRs that were among the top 15 upregulated miRNAs from two Toray 3D-Gene microRNA array-based approaches in comparisons of ESCC case and control plasmas and preoperative and post-ESCC plasmas, respectively. Of the top 15 miRNAs from each of these comparisons, there were a common set of eight miRNAs that were selected; (2) test-scale analyses using qRT–PCR in order to validate the utility of selected candidates from miRNA array-based approaches, comparing the plasma levels of the eight common miRNAs selected in 20 ESCC patients and 10 healthy controls; (3) evaluation of the correlation between each plasma miRNA level and peripheral blood cells in ESCC patients; (4) large-scale analysis of validation of plasma miR-25 levels by comparing 105 preoperative and 20 superficial ESCC patients who underwent curative oesophagectomy and endoscopic resection, respectively, with 50 healthy volunteers; and (5) evaluation of whether plasma miR-25 levels reflect tumour dynamics in the treatment course of ESCC patients ( Figure 1 ).
Selection of plasma miRNA candidates through the integration of two comprehensive miRNA array-based approaches
Novel miRNA candidates in the plasma for cancer detection and tumour monitoring were selected while using two miRNA array-based approaches to compare plasma miRNA levels between ESCC patients and healthy volunteers ( Figure 2A ) and between preoperative and postoperative ESCC patients ( Figure 2B ). Of the 1719 candidate miRNAs analysed, 219 plasma miRNAs were more upregulated in ESCC patients than in healthy volunteers with two-fold changes ( Supplementary Table S1 ), whereas 156 plasma miRNAs were more upregulated in preoperative ESCC patients than in postoperative ESCC patients with two-fold changes ( Supplementary Table S2 ). In order to detect more sensitive biomarkers, we focused on the top 15 miRs in this study ( Figure 2 ). All data were summarised in Supplementary Tables S1 and S2 . Of the 15 highly upregulated miRNAs from each group, eight common miRNAs (miR-15b, 16, 17, 25, 19b, 20a, 20b, and 106a) were selected. Concerning miR-451, which we previously reported in gastric cancer detection and monitoring ( Konishi et al , 2012 ), the origin of high miR-451 level in plasma is currently uncertain in cancers, because miR-451 is known as a tumour-suppressive microRNA and downexpressed in primary cancers. ( Konishi et al , 2012 ). Therefore, in this study, we excluded miR-451 for further analyses, because plasma miR-451 needs considerable investigations and is not still suitable for biomarker in cancer detection and tumour monitoring.
Test-scale analyses comparing plasma levels of eight common miRNAs in ESCC patients and healthy controls
For test-scale analyses, we investigated the plasma levels of the eight miRNAs selected in 20 ESCC patients and 10 health volunteers by quantitative RT–PCR. miR-25 ( P =0.0030) and miR-19b ( P =0.0020) were validated to be more significantly upregulated in ESCC patients than in healthy volunteers ( Figure 3 ). miR-106a ( P =0.0172) and miR-15b ( P =0.0109) were significantly lower in the plasma of ESCC patients than of healthy volunteers. These miRNAs were excluded from further analyses, because their expression patterns were inversely different from those reflected in the array-based results ( Figure 2 ).
Evaluation of the correlation between plasma miRNA levels and peripheral blood cells
Recent reports demonstrated that some circulating miRNAs may be derived from peripheral blood cells ( Pritchard et al , 2012 ; Cheng et al , 2013 ). We determined the correlation between the plasma levels of the miRNAs selected and peripheral blood cells in 20 ESCC patients. We found a significant correlation between the plasma miR-19b levels and concentrations of red blood cells and haemoglobin in the peripheral blood ( Figure 4A ). Considering the influence of secretion of miRNAs from blood cells or haemolysis on the results of clinical application, we excluded miR-19b from further analyses in this study. As no significant correlation was observed between plasma miR-25 levels and any type of blood cell in the peripheral blood of 20 ESCC patients,we did not eliminate this miRNA from further analyses ( Figure 4B ).
Large-scale analysis of validation of plasma miR-25 levels by comparing ESCC patients with healthy volunteers
Next, plasma miR-25 levels were examined on a large scale for our validation study using plasma samples of ESCC patients and healthy volunteers in qRT–PCR assays. The linearity of qRT–PCR was confirmed from concentrations of 1 fmol to 0.0001 fmol of each synthetic miRNA, such as miR-25 ( R 2 =0.9934) between the logarithm of the amount of input miRNA and Ct values ( Supplementary Figure S2 ). Plasma miR-25 was detectable in all samples from 105 consecutive ESCC patients and 50 healthy volunteers. The differential expression of plasma miR-25 in the ESCC patients was compared with that in normal healthy volunteers using a waterfall plot ( Figure 5C ). Plasma miR-25 levels were significantly higher in the ESCC patients than in the healthy volunteers ( P <0.0001; ESCC patients vs healthy volunteers (mean±s.d.): 0.51±0.57 vs 0.19±0.23 amol μ l −1 ) ( Figure 5C ). No significant correlation was observed between the plasma miR-25 levels and any type of blood cell in the peripheral blood of the 105 ESCC patients ( Supplementary Figure S3 ). Table 1 shows the association between the plasma miR-25 levels and clinicopathological factors in the 105 consecutive ESCC patients. A high plasma miR-25 level was shown to be significantly associated with the absence of lymphatic invasion ( P =0.0110) and early tumour stage in ESCC ( P =0.0115).
Further study on clinical application of plasma miR-25 in superficial ESCC patients undergoing endoscopic resection
To further evaluate the possibility of clinical application of plasma miR-25, plasma miR-25 levels in 20 consecutive superficial ESCC patients who underwent curative endoscopic resection (m2-sm1, R0) and 50 healthy volunteers were examined by real-time PCR ( Figure 6A ). The plasma miR-25 levels were significantly higher in the superficial ESCC patients than in the healthy volunteers (mean±s.d.: 1.82±0.54 vs 0.19±0.23, P <0.0001). Figure 6B shows that the value for the AUC for the plasma miR-25 analysis was 0.8555. To detect any cutoff points that could differentiate cancer patients from healthy volunteers, we utilised the AUC with the Youden index ( Akobeng, 2007 ). An optimal cutoff point was indicated at 0.32 amol μ l −1 , with a sensitivity of 85.0% and a specificity of 86.0%. Indeed, the sensitivity of plasma miR-25 for superficial ESCC detection was much higher than that of conventional tumour markers, including SCC, CEA, and CYFRA, at our institute (pT0-1; SCC, CEA, and CYFRA: 25.0, 21.1, and 12.5%, respectively) (details not shown). Our results provided evidence that plasma miR-25 levels can be used to distinguish early ESCC patients from healthy volunteers to a clinically satisfactory degree in comparison with conventional tumour markers.
Evaluation to determine whether plasma miR-25 levels reflect tumour dynamics in the treatment course of ESCC patients
To validate whether plasma miR-25 levels reflect tumour dynamics, we first confirmed the higher miR-25 expression in primary ESCC tissues and ESCC cell lines. miR-25 expression in 10 ESCC tissues and 7 normal oesophageal tissues was determined by qRT–PCR. Moreover, the human ESCC cell lines TE-2, TE-9, TE-13, and KYSE70, and a human fibroblast cell line WI-38 were evaluated by qRT–PCR. The results are shown in Figure 5A and B after normalisation to control U6 expression. miR-25 levels were significantly higher in ESCC tissues than in normal ESCC tissues ( P =0.0157). Furthermore, the miR-25 levels were significantly higher in the ESCC cell lines than in the fibroblast cell line and the normal oesophageal tissues ( P =0.0093). Consequently, miR-18a expression was shown to be significantly increased in most ESCC tissues and cell lines but not in normal tissues and fibroblasts. Second, the miR-25 level was analysed in paired preoperative and postoperative plasma samples from ESCC patients who underwent curative oesophagectomy and endoscopic resection. As shown in the array-based approach ( Figure 2B ), plasma miR-25 levels were shown to be significantly reduced in samples after oesophagectomy ( P =0.0001) ( Figure 7A ). In some patients from whom paired samples were collected after oesophagectomy and after recurrences, a significant re-elevation in the plasma miR-25 levels was found at recurrence ( P =0.0145) ( Figure 7B ). These findings indicated that the plasma miR-25 level might reflect tumour dynamics in the treatment course of ESCC patients.
Discussion
During the course of a programme of genome-wide miRNA profiling in the plasma of ESCC patients using high-resolution miRNA arrays, we identified a novel plasma miR-25. The plasma expression of this miRNA was significantly higher in preoperative ESCC patients and in pretreatment superficial ESCC patients undergoing endoscopic resection than in postoperative ESCC patients and healthy volunteers. Moreover, plasma miR-25 expression was independent of the blood cell status and reflects the tumour dynamics of preoperation and postoperation and recurrences. Our results provide evidence that plasma miR-25 levels contribute to cancer detection and can be used to monitor ESCC patients to a clinically satisfactory degree in comparison with conventional tumour markers.
During the past decade, non-coding RNAs, so-called miRNAs, have been demonstrated to regulate gene expression by targeting mRNAs for translational repression or cleavage. Moreover, miRNAs have emerged as integral components of the oncogenic and tumour-suppressor network, regulating almost all cellular processes altered during tumour development and may provide new therapeutic strategies such as biomarkers and therapeutic targets for cancers ( He et al , 2005 ; Lu et al , 2005 ; Calin and Croce, 2006 ; He et al , 2007 ). Furthermore, miRNAs have been shown to be present in a remarkably stable form in plasma/serum, and the expression level of serum/plasma miRNAs is reproducible and consistent among individuals ( Chen et al , 2008 ; Mitchell et al , 2008 ). In fact, a number of reports have suggested the presence of circulating miRNAs and their potential use as novel biomarkers for cancers, such as prostate cancer ( Mitchell et al , 2008 ), leukaemia ( Lawrie et al , 2008 ), oral cancer ( Wong et al , 2008 ), pancreatic cancer ( Wang et al , 2009 ; Morimura et al , 2011 ; Kawaguchi et al , 2013 ), colorectal cancer ( Ng et al , 2009 ), lung cancer ( Hu et al , 2010 ), breast cancer ( Heneghan et al , 2010 ), and gastric cancer ( Tsujiura et al , 2010 ; Konishi et al , 2012 ).
In ESCC, there have been only a few reports, including three reports from our research group, on the role of circulating miRNAs in plasma/serum patients with ESCC ( Zhang et al , 2010 ; Komatsu et al , 2011 , 2012 ; Kurashige et al , 2012 ; Hirajima et al , 2013 ; Takeshita et al , 2013 ). However, these reports did not always indicate that these reported miRNAs were the only candidate biomarkers for ESCC. Therefore more sensitive and promising candidate miRNAs for screening cancer, monitoring the tumour status, and predicting prognosis need to be found in clinical settings. There have been only two specific reports about serum miRNA using the comprehensive array-based approach. Namely, Zhang et al (2010) investigated the serum miRNA profiles in ESCC patients using Solexa sequencing. Among the 25 selected candidate miRNAs analysed, they identified 7 serum miRNAs (miR-10a, 22, 100, 148b, 223, 133a, and 127-3p) as ESCC biomarkers ( Zhang et al , 2010 ). Moreover, Takeshita et al (2013) examined serum miRNA profiles using Agilent human miRNA microarrays to compare the serum miRNA levels in ESCC patients with the levels in healthy controls. Thus miR-1246 was the most markedly elevated miRNA in ESCC patients and was proved to be an independent poor prognostic factor ( Takeshita et al , 2013 ). However, there have been no reported miRNAs in the plasma of ESCC patients that had been identified by the comprehensive array-based approach. Previous studies about miRNA profiles revealed that the majority of circulating miRNAs were co-fractionated with plasma protein complexes ( Arroyo et al , 2011 ). Moreover, plasma might include more abundant protein levels, including cogulant-related proteins, than serum. Indeed, the detected candidate miRNAs were considerably different, because the profiles of cancer-associated miRNAs might be different between plasma and serum ( Takeshita et al , 2013 ). These findings prompted us to further search for novel cancer-associated miRNAs in plasma through a comprehensive miRNA microarray-based approach.
In this study, we finally identified a novel ESCC screening and monitoring plasma miRNA, miR-25, which is included in the miR-106b-25 cluster comprising miR-106b, miR-93, and miR-25 ( Yu et al , 2006 ) and is located within the intron13 of MCM7 gene locus at 7q23.1. The region for these miRNAs was reported to be frequently overexpressed/amplified in ESCC tissue and cell lines ( Shinomiya et al , 1999 ; Guo et al , 2008 ; Sakai et al , 2010 ; Zhu et al , 2011 ; Xu et al , 2012 ). This genomic aberration of the miR-106b-25 cluster was also accumulated in various other cancers, including gastric cancer ( Petrocca et al , 2008 ), hepatocellular carcinoma ( Li et al , 2009 ), prostate cancer ( Poliseno et al , 2010 ), multiple myeloma ( Pichiorri et al , 2008 ), ovarian cancer ( Zhang et al , 2012 ), and breast cancer ( Wu et al , 2012 ). Furthermore, several recent studies have demonstrated that the miR-106b-25 cluster and its individual miRNAs have pro-oncogenic functions, including mediating pro-proliferative and antiapoptotic phenotypes ( Ren et al , 2006 ; Petrocca et al , 2008 ; Kan et al , 2009 ; Li et al , 2009 ; Poliseno et al , 2010 ; Martens-Uzunova et al , 2011 ). Some of the identified crucial functions of miR-106b-25 are the inhibition of tumour-suppressor genes such as PTEN ( Poliseno et al , 2010 ), p21 / WAF1 and Bim ( Petrocca et al , 2008 ; Kan et al , 2009 ; Li et al , 2009 ), TP53 ( Kumar et al , 2011 ), CDH1 ( Xu et al , 2012 ), and DR4 ( Razumilava et al , 2012 ) in several cancers and the activation of E2F1 , which leads to impaired TGF-β signalling and to proliferation, dysregulated cell cycling, and increased invasiveness in HNSCC ( Hui et al , 2010 ). Furthermore, these oncogenic miRNAs in the miR-106b-25 cluster have been shown to be upregulated in cancer stromal tissues compared with normal stroma ( Nishida et al , 2012 ). These findings strongly suggested that miR-25 may have a pivotal role in carcinogenesis and the development of tumours and that upregulation of plasma miR-25 might be associated with the molecular behaviours of ESCC. Indeed, a high plasma miR-25 level was proved to be significantly associated with early tumour stage in ESCC ( P =0.0115), and in superficial ESCC patients undergoing endoscopic resection, the value for the AUC, which was used to assess the feasibility of using plasma miRNA levels as a diagnostic tool for the early detection of ESCC, was great (0.8555). This suggests that miR-25 has an important role in the early phase of cancer development. Thus plasma miR-25 levels may be a useful biomarker for the early detection of ESCC.
To further evaluate the clinical possibility of miR-25 for monitoring tumour dynamics, we investigated whether plasma miR-25 levels could reflect tumour dynamics in ESCC by three different analyses. The first analysis was the confirmation of comparative higher miR-25 expression in primary ESCC tissues and ESCC cell lines than in normal oesophageal tissues and fibroblast cells. The second analysis was to confirm that the miR-25 levels were significantly reduced after curative oesophagectomy and endoscopic resection in patients with high preoperative plasma miR-25 levels ( Figure 7A ). The third analysis was to determine that the significant re-elevation in the plasma miR-25 levels was found at recurrences after curative oesophagectomy or endoscopic resection ( Figure 7B ). These findings were similar to those of our previous reports ( Tsujiura et al , 2010 ; Komatsu et al , 2011 ; Morimura et al , 2011 ; Konishi et al , 2012 ; Hirajima et al , 2013 ; Kawaguchi et al , 2013 ) and clearly demonstrated that plasma miR-25 levels reflected tumour dynamics and may be available as a new plasma biomarker for monitoring the tumour status of ESCC patients.
Recently, Pritchard and his colleagues reported a caution in a cancer biomarker study of circulating miRNAs because the circulating miRNAs might have been derived from peripheral blood cells ( Pritchard et al , 2012 ; Cheng et al , 2013 ). The specific miRNAs associated with haemolysis and the secretion of miRNAs from blood cells might affect their plasma levels in clinical application. Therefore we evaluated the correlation between both miR-25 and miR-19 levels in plasma and peripheral blood cells in both 20 ESCC patients as a test study and in 105 consecutive ESCC patients as a validation cohort study. A significant association was observed between the plasma miR-19 levels and concentrations of red blood cells and haemoglobin in the peripheral blood ( Figure 4A ). However, as for miR-25, no association was found between them in both the 20 ESCC patients as a test study and the 105 consecutive ESCC patients as a validation cohort study ( Figure 4B , Supplementary Figure S3 ). These results might exclude the possibility that the secretion of miRNAs from blood cells or haemolysis affected the miR-25 level in clinical settings. However, because secretion, kinetics, and metabolism in plasma miRNAs have not been clearly elucidated, this issue is currently under evaluation.
Taken together, through the integration of two comprehensive miRNA array-based approaches, we clearly demonstrated that plasma miR-25 levels may potentially be useful for screening cancer and monitoring tumour dynamics in ESCC patients. Nevertheless, many issues must be addressed before these findings can be translated into a clinically useful, non-invasive screening strategy for ESCC patients. Therefore we will prospectively confirm the usefulness of plasma miR-25 in a large number of patients. Furthermore, we believe that more sensitive plasma miRNA biomarkers could be identified by different methods such as a recent next-generation sequence or digital PCR-based approach and could translate circulating miRNAs into the clinical setting. These strategies are currently under evaluation, and we will report in the near future.
Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)
This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License.
Adélaide J Monges G Dérdérian C Seitz JF Birnbaum D 1995 Oesophageal cancer and amplification of the human cyclin D gene CCND1/PRAD1 Br J Cancer 71 64 68 7819051 10.1038/bjc.1995.13 PMC2033462
Akobeng AK 2007 Understanding diagnostic tests 3: receiver operating characteristic curves Acta Paediatr 96 644 647 17376185 10.1111/j.1651-2227.2006.00178.x
Arroyo JD Chevillet JR Kroh EM Ruf IK Pritchard CC Gibson DF Mitchell PS Bennett CF Pogosova-Agadjanyan EL Stirewalt DL Tait JF Tewari M 2011 Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma Proc Natl Acad Sci USA 108 5003 5008 21383194 10.1073/pnas.1019055108 PMC3064324
Calin GA Croce CM 2006 MicroRNA signatures in human cancers Nat Rev Cancer 6 857 866 17060945 10.1038/nrc1997
Chen X Ba Y Ma L Cai X Yin Y Wang K Guo J Zhang Y Chen J Guo X Li Q Li X Wang W Wang J Jiang X Xiang Y Xu C Zheng P Zhang J Li R Zhang H Shang X Gong T Ning G Zen K Zhang CY 2008 Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases Cell Res 18 997 1006 18766170 10.1038/cr.2008.282
Cheng HH Yi HS Kim Y Kroh EM Chien JW Eaton KD Goodman MT Tait JF Tewari M Pritchard CC 2013 Plasma processing conditions substantially influence circulating microRNA biomarker levels PLoS One 8 e64795 23762257 10.1371/journal.pone.0064795 PMC3676411
Cocucci E Racchetti G Meldolesi J 2009 Shedding microvesicles: artefacts no more Trends Cell Biol 19 43 41 19144520 10.1016/j.tcb.2008.11.003
Fang WK Liao LD Li LY Xie YM Xu XE Zhao WJ Wu JY Zhu MX Wu ZY Du ZP Wu BL Xie D Guo MZ Xu LY Li EM 2013 Down-regulated desmocollin-2 promotes cell aggressiveness through redistributing adherens junctions and activating beta-catenin signalling in oesophageal squamous cell carcinoma J Pathol 231 257 270 23836524 10.1002/path.4236
Filipowicz W Bhattacharyya SN Sonenberg N 2008 Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight Nat Rev Genet 9 102 114 18197166 10.1038/nrg2290
Giovannetti E van der Velde A Funel N Vasile E Perrone V Leon LG De Lio N Avan A Caponi S Pollina LE Gallá V Sudo H Falcone A Campani D Boggi U Peters GJ 2012 High-throughput microRNA (miRNAs) arrays unravel the prognostic role of MiR-211 in pancreatic cancer PLoS One 7 e49145 23155457 10.1371/journal.pone.0049145 PMC3498320
Gratas C Tohma Y Barnas C Taniere P Hainaut P Ohgaki H 1998 Up-regulation of Fas (APO-1/CD95) ligand and down-regulation of Fas expression in human esophageal cancer Cancer Res 58 2057 2062 9605741
Guo Y Chen Z Zhang L Zhou F Shi S Feng X Li B Meng X Ma X Luo M Shao K Li N Qiu B Mitchelson K Cheng J He J 2008 Distinctive microRNA profiles relating to patient survival in esophageal squamous cell carcinoma Cancer Res 68 26 33 18172293 10.1158/0008-5472.CAN-06-4418
Hasselmann DO Rappl G Tilgen W Reinhold U 2001 Extracellular tyrosinase mRNA within apoptotic bodies is protected from degradation in human serum Clin Chem 47 1488 1489 11468248
He L He X Lim LP de Stanchina E Xuan Z Liang Y Xue W Zender L Magnus J Ridzon D Jackson AL Linsley PS Chen C Lowe SW Cleary MA Hannon GJ 2007 A microRNA component of the p53 tumour suppressor network Nature 447 1130 1134 17554337 10.1038/nature05939 PMC4590999
He L Thomson JM Hemann MT Hernando-Monge E Mu D Goodson S Powers S Cordon-Cardo C Lowe SW Hannon GJ Hammond SM 2005 A microRNA polycistron as a potential human oncogene Nature 435 828 833 15944707 10.1038/nature03552 PMC4599349
Heneghan HM Miller N Lowery AJ Sweeney KJ Newell J Kerin MJ 2010 Circulating microRNAs as novel minimally invasive biomarkers for breast cancer Ann Surg 251 499 505 20134314 10.1097/SLA.0b013e3181cc939f
Hirajima S Komatsu S Ichikawa D Takeshita H Konishi H Shiozaki A Morimura R Tsujiura M Nagata H Kawaguchi T Arita T Kubota T Fujiwara H Okamoto K Otsuji E 2013 Clinical impact of circulating miR-18a in plasma of patients with oesophageal squamous cell carcinoma Br J Cancer 108 1822 1829 23579215 10.1038/bjc.2013.148 PMC3658511
Hiyama T Yoshihara M Tanaka S Chayama K 2007 Genetic polymorphisms and esophageal cancer risk Int J Cancer 121 1643 1658 17674367 10.1002/ijc.23044
Hollstein MC Metcalf RA Welsh JA Montesano R Harris CC 1990 Frequent mutation of the p53 gene in human esophageal cancer Proc Natl Acad Sci USA 87 9958 9961 2263646 10.1073/pnas.87.24.9958 PMC55293
Hu Z Chen X Zhao Y Tian T Jin G Shu Y Chen Y Xu L Zen K Zhang C Shen H 2010 Serum microRNA signatures identified in a genome-wide serum microRNA expression profiling predict survival of non-small-cell lung cancer J Clin Oncol 28 1721 1726 20194856 10.1200/JCO.2009.24.9342
Hui AB Lenarduzzi M Krushel T Waldron L Pintilie M Shi W Perez-Ordonez B Jurisica I O'Sullivan B Waldron J Gullane P Cummings B Liu FF 2010 Comprehensive MicroRNA profiling for head and neck squamous cell carcinomas Clin Cancer Res 16 1129 1139 20145181 10.1158/1078-0432.CCR-09-2166
Ichikawa D Komatsu S Konishi H Otsuji E 2012 Circulating microRNA in digestive tract cancers Gastroenterology 142 1074 1078 22433392 10.1053/j.gastro.2012.03.008
Jemal A Bray F Center MM Ferlay J Ward E Forman D 2011 Global cancer statistics CA Cancer J Clin 61 69 90 21296855 10.3322/caac.20107
Kan T Sato F Ito T Matsumura N David S Cheng Y Agarwal R Paun BC Jin Z Olaru AV Selaru FM Hamilton JP Yang J Abraham JM Mori Y Meltzer SJ 2009 The miR-106b-25 polycistron, activated by genomic amplification, functions as an oncogene by suppressing p21 and Bim Gastroenterology 136 1689 1700 19422085 10.1053/j.gastro.2009.02.002 PMC2887605
Kawaguchi T Komatsu S Ichikawa D Morimura R Tsujiura M Konishi H Takeshita H Nagata H Arita T Hirajima S Shiozaki A Ikoma H Okamoto K Ochiai T Taniguchi H Otsuji E 2013 Clinical impact of circulating miR-221 in plasma of patients with pancreatic cancer Br J Cancer 108 361 369 23329235 10.1038/bjc.2012.546 PMC3566805
Komatsu S Ichikawa D Takeshita H Konishi H Nagata H Hirajima S Kawaguchi T Arita T Shiozaki A Fujiwara H Okamoto K Otsuji E 2012 Prognostic impact of circulating miR-21 and miR-375 in plasma of patients with esophageal squamous cell carcinoma Expert Opin Biol Ther 12 (Suppl 1 S53 S59 22519435 10.1517/14712598.2012.681373
Komatsu S Ichikawa D Takeshita H Tsujiura M Morimura R Nagata H Kosuga T Iitaka D Konishi H Shiozaki A Fujiwara H Okamoto K Otsuji E 2011 Circulating microRNAs in plasma of patients with oesophageal squamous cell carcinoma Br J Cancer 105 104 111 21673684 10.1038/bjc.2011.198 PMC3137413
Konishi H Ichikawa D Komatsu S Shiozaki A Tsujiura M Takeshita H Morimura R Nagata H Arita T Kawaguchi T Hirashima S Fujiwara H Okamoto K Otsuji E 2012 Detection of gastric cancer-associated microRNAs on microRNA microarray comparing pre- and post-operative plasma Br J Cancer 106 740 747 22262318 10.1038/bjc.2011.588 PMC3322946
Kosaka N Iguchi H Yoshioka Y Takeshita F Matsuki Y Ochiya T 2010 Secretory mechanisms and intercellular transfer of microRNAs in living cells J Biol Chem 285 17442 17452 20353945 10.1074/jbc.M110.107821 PMC2878508
Kosugi S Nishimaki T Kanda T Nakagawa S Ohashi M Hatakeyama K 2004 Clinical significance of serum carcinoembryonic antigen, carbohydrate antigen 19-9, and squamous cell carcinoma antigen levels in esophageal cancer patients World J Surg 28 680 685 15383868 10.1007/s00268-004-6865-y
Kumar M Lu Z Takwi AA Chen W Callander NS Ramos KS Young KH Li Y 2011 Negative regulation of the tumor suppressor p53 gene by microRNAs Oncogene 30 843 853 20935678 10.1038/onc.2010.457 PMC3021102
Kurashige J Kamohara H Watanabe M Tanaka Y Kinoshita K Saito S Hiyoshi Y Iwatsuki M Baba Y Baba H 2012 Serum microRNA-21 is a novel biomarker in patients with esophageal squamous cell carcinoma J Surg Oncol 106 188 192 22354855 10.1002/jso.23064
Lawrie CH Gal S Dunlop HM Pushkaran B Liggins AP Pulford K Banham AH Pezzella F Boultwood J Wainscoat JS Hatton CS Harris AL 2008 Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma Br J Haematol 141 672 675 18318758 10.1111/j.1365-2141.2008.07077.x
Lee RC Feinbaum RL Ambros V 1993 The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 Cell 75 843 854 8252621 10.1016/0092-8674(93)90529-y
Lewis BP Burge CB Bartel DP 2005 Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets Cell 120 15 20 15652477 10.1016/j.cell.2004.12.035
Li Y Tan W Neo TW Aung MO Wasser S Lim SG Tan TM 2009 Role of the miR-106b-25 microRNA cluster in hepatocellular carcinoma Cancer Sci 100 1234 1242 19486339 10.1111/j.1349-7006.2009.01164.x
Livak KJ Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods 25 402 408 11846609 10.1006/meth.2001.1262
Lu J Getz G Miska EA Alvarez-Saavedra E Lamb J Peck D Sweet-Cordero A Ebert BL Mak RH Ferrando AA Downing JR Jacks T Horvitz HR Golub TR 2005 MicroRNA expression profiles classify human cancers Nature 435 834 838 15944708 10.1038/nature03702
Martens-Uzunova ES Jalava SE Dits NF van Leenders GJ Møller S Trapman J Bangma CH Litman T Visakorpi T Jenster G 2011 Diagnostic and prognostic signatures from the small non-coding RNA transcriptome in prostate cancer Oncogene 31 978 991 21765474 10.1038/onc.2011.304
Meng XY Zhu ST Zong Y Wang YJ Li P Zhang ST 2011 Promoter hypermethylation of cyclooxygenase-2 gene in esophageal squamous cell carcinoma Dis Esophagus 24 444 449 21166741 10.1111/j.1442-2050.2010.01159.x
Mitchell PS Parkin RK Kroh EM Fritz BR Wyman SK Pogosova-Agadjanyan EL Peterson A Noteboom J O'Briant KC Allen A Lin DW Urban N Drescher CW Knudsen BS Stirewalt DL Gentleman R Vessella RL Nelson PS Martin DB Tewari M 2008 Circulating microRNAs as stable blood-based markers for cancer detection Proc Natl Acad Sci USA 105 10513 10518 18663219 10.1073/pnas.0804549105 PMC2492472
Morimura R Komatsu S Ichikawa D Takeshita H Tujiura M Nagata H Konishi H Shiozaki A Ikoma H Okamoto K Ochiai T Taniguchi H Otsuji E 2011 Novel diagnostic value of circulating miR-18a in plasma of patients with pancreatic cancer Br J Cancer 11 1733 1740 22045190 10.1038/bjc.2011.453 PMC3242609
Mroczko B Kozłowski M Groblewska M Łukaszewicz M Nikliński J Jelski W Laudański J Chyczewski L Szmitkowski M 2008 The diagnostic value of the measurement of matrix metalloproteinase 9 (MMP-9), squamous cell cancer antigen (SCC) and carcinoembryonic antigen (CEA) in the sera of esophageal cancer patients Clin Chim Acta 389 61 66 18155162 10.1016/j.cca.2007.11.023
Nagino K Nomura O Takii Y Myomoto A Ichikawa M Nakamura F Higasa M Akiyama H Nobumasa H Shiojima S Tsujimoto G 2006 Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification J Biochem 139 697 703 16672270 10.1093/jb/mvj086
Ng EK Chong WW Jin H Lam EK Shin VY Yu J Poon TC Ng SS Sung JJ 2009 Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening Gut 58 1375 1381 19201770 10.1136/gut.2008.167817
Nishida N Nagahara M Sato T Mimori K Sudo T Tanaka F Shibata K Ishii H Sugihara K Doki Y Mori M 2012 Microarray analysis of colorectal cancer stromal tissue reveals upregulation of two oncogenic miRNA clusters Clin Cancer Res 18 3054 3070 22452939 10.1158/1078-0432.CCR-11-1078
Petrocca F Visone R Onelli MR Shah MH Nicoloso MS de Martino I Iliopoulos D Pilozzi E Liu CG Negrini M Cavazzini L Volinia S Alder H Ruco LP Baldassarre G Croce CM Vecchione A 2008 E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer Cancer Cell 13 272 286 18328430 10.1016/j.ccr.2008.02.013
Pichiorri F Suh SS Ladetto M Kuehl M Palumbo T Drandi D Taccioli C Zanesi N Alder H Hagan JP Munker R Volinia S Boccadoro M Garzon R Palumbo A Aqeilan RI Croce CM 2008 MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis Proc Natl Acad Sci USA 105 12885 12890 18728182 10.1073/pnas.0806202105 PMC2529070
Poliseno L Salmena L Riccardi L Fornari A Song MS Hobbs RM Sportoletti P Varmeh S Egia A Fedele G Rameh L Loda M Pandolfi PP 2010 Identification of the miR-106b∼25 microRNA cluster as a proto-oncogenic PTEN-targeting intron that cooperates with its host gene MCM7 in transformation Sci Signal 3 ra29 Erratum in: Sci Signal. 2010; 3 (123):er6. 20388916 10.1126/scisignal.2000594 PMC2982149
Pritchard CC Kroh E Wood B Arroyo JD Dougherty KJ Miyaji MM Tait JF Tewari M 2012 Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies Cancer Prev Res (Phila) 5 492 497 22158052 10.1158/1940-6207.CAPR-11-0370 PMC4186243
Razumilava N Bronk SF Smoot RL Fingas CD Werneburg NW Roberts LR Mott JL 2012 miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma Hepatology 55 465 475 21953056 10.1002/hep.24698 PMC3268937
Rechavi O Erlich Y Amram H Flomenblit L Karginov FV Goldstein I Hannon GJ Kloog Y 2009 Cell contact-dependent acquisition of cellular and viral nonautonomously encoded small RNAs Genes Dev 23 1971 1979 19684116 10.1101/gad.1789609 PMC2725935
Ren B Yu G Tseng GC Cieply K Gavel T Nelson J Michalopoulos G Yu YP Luo JH 2006 MCM7 amplification and overexpression are associated with prostate cancer progression Oncogene 25 1090 1098 16247466 10.1038/sj.onc.1209134
Sakai N Kajiyama Y Iwanuma Y Tomita N Amano T Isayama F Ouchi K Tsurumaru M 2010 Study of abnormal chromosome regions in esophageal squamous cell carcinoma by comparative genomic hybridization: relationship of lymph node metastasis and distant metastasis to selected abnormal regions Dis Esophagus 23 415 421 19930403 10.1111/j.1442-2050.2009.01026.x
Shi HY Lv FJ Zhu ST Wang QG Zhang ST 2011 Dual inhibition of 5-LOX and COX-2 suppresses esophageal squamous cell carcinoma Cancer Lett 309 19 26 21652147 10.1016/j.canlet.2011.05.010
Shinomiya T Mori T Ariyama Y Sakabe T Fukuda Y Murakami Y Nakamura Y Inazawa J 1999 Comparative genomic hybridization of squamous cell carcinoma of the esophagus: the possible involvement of the DPI gene in the 13q34 amplicon Genes Chromosomes Cancer 24 337 344 10092132
Skog J Würdinger T van Rijn S Meijer DH Gainche L Sena-Esteves M Curry WT Jr Carter BS Krichevsky AM Breakefield XO 2008 Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers Nat Cell Biol 10 1470 1476 19011622 10.1038/ncb1800 PMC3423894
Sobin LH Gospodarowicz MK Wittekind C 2009 TNM Classification of Malignant Tumors 7th edn. Wiley-liss: New York, NY, USA
Takeshita N Hoshino I Mori M Akutsu Y Hanari N Yoneyama Y Ikeda N Isozaki Y Maruyama T Akanuma N Komatsu A Jitsukawa M Matsubara H 2013 Serum microRNA expression profile: miR-1246 as a novel diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma Br J Cancer 108 644 652 23361059 10.1038/bjc.2013.8 PMC3593570
Tsujiura M Ichikawa D Komatsu S Shiozaki A Takeshita H Kosuga T Konishi H Morimura R Deguchi K Fujiwara H Okamoto K Otsuji E 2010 Circulating microRNAs in plasma of patients with gastric cancers Br J Cancer 102 1174 1179 20234369 10.1038/sj.bjc.6605608 PMC2853097
Valadi H Ekstrom K Bossios A Sjostrand M Lee JJ Lotvall JO 2007 Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells Nat Cell Biol 9 654 659 17486113 10.1038/ncb1596
Vickers KC Palmisano BT Shoucri BM Shamburek RD Remaley AT 2011 MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins Nat Cell Biol 13 423 433 21423178 10.1038/ncb2210 PMC3074610
Wang J Chen J Chang P LeBlanc A Li D Abbruzzesse JL Frazier ML Killary AM Sen S 2009 MicroRNAs in plasma of pancreatic ductal adenocarcinoma patients as novel blood-based biomarkers of disease Cancer Prev Res (Phila) 2 807 813 19723895 10.1158/1940-6207.CAPR-09-0094 PMC5859193
Wong TS Liu XB Wong BY Ng RW Yuen AP Wei WI 2008 Mature miR-184 as potential oncogenic microRNA of squamous cell carcinoma of tongue Clin Cancer Res 14 2588 2592 18451220 10.1158/1078-0432.CCR-07-0666
Wu Q Wang C Lu Z Guo L Ge Q 2012 Analysis of serum genome-wide microRNAs for breast cancer detection Clin Chim Acta 413 1058 1065 22387599 10.1016/j.cca.2012.02.016
Xu X Chen Z Zhao X Wang J Ding D Wang Z Tan F Tan X Zhou F Sun J Sun N Gao Y Shao K Li N Qiu B He J 2012 MicroRNA-25 promotes cell migration and invasion in esophageal squamous cell carcinoma Biochem Biophys Res Commun 421 640 645 22450326 10.1016/j.bbrc.2012.03.048
Yu J Wang F Yang GH Wang FL Ma YN Du ZW Zhang JW 2006 Human microRNA clusters: genomic organization and expression profile in leukemia cell lines Biochem Biophys Res Commun 349 59 68 16934749 10.1016/j.bbrc.2006.07.207
Zhang C Wang C Chen X Yang C Li K Wang J Dai J Hu Z Zhou X Chen L Zhang Y Li Y Qiu H Xing J Liang Z Ren B Yang C Zen K Zhang CY 2010 Expression profile of microRNAs in serum: a fingerprint for esophageal squamous cell carcinoma Clin Chem 56 1871 1879 20943850 10.1373/clinchem.2010.147553
Zhang H Zuo Z Lu X Wang L Wang H Zhu Z 2012 MiR-25 regulates apoptosis by targeting Bim in human ovarian cancer Oncol Rep 27 594 598 22076535 10.3892/or.2011.1530
Zhu H Fan GC 2011 Extracellular/circulating microRNAs and their potential role in cardiovascular disease Am J Cardiovasc Dis 2 138 149 22059153 PMC3207246
Zhu L Yan W Rodriguez-Canales J Rosenberg AM Hu N Goldstein AM Taylor PR Erickson HS Emmert-Buck MR Tangrea MA 2011 MicroRNA analysis of microdissected normal squamous esophageal epithelium and tumor cells Am J Cancer Res 1 574 584 21796275 PMC3142940
Supplementary Material
Click here for additional data file.
Click here for additional data file.
Click here for additional data file.
Click here for additional data file.
Click here for additional data file.
Click here for additional data file.