Sutang Guo, Department of Molecular Biology, Shanxi Cancer Hospital and Institute, Taiyuan, Shanxi 030013, China. E-mail: sutangguo@aliyun.com
Chunyan Wang, Department of Molecular Biology, Department of Blood Transfusion, Shanxi Cancer Hospital and Institute, Taiyuan, Shanxi 030013, China. E-mail: c3213258@uon.edu.au
This paper was designed to explore the value of miRNAs as diagnostic biomarkers that may facilitate the early detection of esophageal squamous cell carcinoma (ESCC). Plasma miRNA profiles were defined via an array-based approach using samples from ESCC patients and healthy controls (n=5 each). Differentially expressed miRNAs in these samples were validated via qPCR in ESCC patients (n=96) and healthy controls (n=51), and the relationship between ESCC patient plasma miR-1260b and miR-720 levels and clinicopathological characteristics were additionally examined. In total, 12 plasma miRNAs that were differentially expressed between ESCC patients and healthy controls were identified via miRNA. Six of these miRNAs were subsequently validated, revealing that both miR-1260b and miR-720 were significantly differentially abundant in ESCC patients and controls, with miR-1260b being significantly upregulated in ESCC patients relative to controls (2.24, 1.41 respectively, P<0.001), while the opposite was observed with respect to miR-720 (0.66, 2.27 respectively, P=0.001). The use of both miR-720 and miR-1260b as a combined diagnostic tool was highly efficacious, yielding an AUC of 0.814, a sensitivity of 86.3%, and a specificity of 73.2% as a means of detecting ESCC patients. Elevated plasma miR-1260b level was also associated with a poorer patient prognosis when compared to patients with a low plasma miRNA level (P=0.021). This study has successfully developed a plasma miRNA biomarker signature of ESCC that may offer value as a diagnostic or prognostic tool when evaluating patients with ESCC.
Esophageal squamous cell carcinoma (ESCC) is a common tumor of the digestive tract that is the sixth deadliest cancer type globally[1-2], representing the predominant histologic esophageal cancer subtype observed in China and other regions of East Asia[3-4]. While advances in the treatment of ESCC through surgery and combination chemoradiotherapy have provided patients with additional options, the 5-year postoperative survival rate remains poor at just 20%–30%[5-7]. There is therefore an urgent need for the identification of novel biomarkers that can facilitate more reliable ESCC patient diagnoses, prognostic evaluation, and/or treatment.
MicroRNAs (miRNAs) are small (20–22 nucleotides) RNAs that lack coding potential, yet are able to facilitate the degradation or translational repression of target mRNAs by binding to complementary sequences in their 3'-untranslated region (UTR)[8-9]. The dysregulation of miRNAs is a hallmark of many cancer types, wherein they can function to promote or suppress tumor growth depending on their specific regulatory targets[10]. Recent work suggests that large quantities of miRNAs are stably present within human serum, such that cancer and other diseases can alter these circulating miRNA profiles in affected patients even when the sources of these miRNAs are not immediately clear[11-12].
The aberrant expression of many different miRNAs is relevant to ESCC progression and patient prognosis[13-18], highlighting these miRNAs as promising biomarkers in the context of this cancer type.
In the present study, a microarray-based approach was employed to identify miRNAs that were differentially abundant in the plasma of ESCC patients relative to healthy controls, with reverse transcription-qPCR then being used to validate these microarray results. The overall goal of this approach was to explore the efficacy of plasma miRNAs as biomarkers for the evaluation of ESCC patients.
SUBJECTS AND METHODS
Patients and samples
All patients participating in this study were enrolled between July 2010 and December 2012 at the Shanxi Province Cancer Hospital. Samples of venous blood were collected from patients prior to any surgical, chemotherapeutic, or radiotherapeutic treatment. In total, plasma samples from 5 ESCC patients and 5 healthy controls were used for microarray analyses, while qPCR validation assays utilized additional samples from 96 ESCC patients and 51 healthy controls. Clinicopathologic features of the 96 ESCC patients are shown as follows: the median age for all patients was 59 years old with a range of 41 to 79 years; males predominated (71%); around half the patients were grade 3 (55/96) and stage II (42/96). The Ethics Committee of Shanxi Province Cancer Hospital approved this study (2020 No.11), and all patients provided written informed consent to participate.
Sample processing and miRNA isolation
Samples of venous blood were spun for 10 minutes at 1000 g within 2 hours following collection. Supernatant plasma samples were then spun for an additional 10 minutes at 12 000 g to remove remaining cellular debris, and were stored at −80 ℃ prior to subsequent analysis.
Total RNA was extracted from plasma samples (200 μL) using a miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) based on provided directions. Prior to RNA extraction, synthetic Caenorhabditis Elegans miR-39 (Ce-miR-39) was added to each plasma sample as an internal control, with samples being eluted with 14 μL of nuclease-free water. RNA samples were then used to prepare cDNA with a miScript II RT Kit (Qiagen) based on provided directions.
miRNA array
The miScript SYBR Green PCR Kit and the Human Serum & Plasma miRNA PCR Array (MIHS-3106ZE, Qiagen) were used to profile plasma levels of 384 different miRNAs in selected patient samples. Array data analysis was then performed with an online application (http://pcrdataanalysis.sabiosciences.com/mirna/arrayanalysis.php), which automatically calculated all △△Ct fold change. Those miRNAs that were differentially expressed in plasma samples from ESCC patients and healthy controls were then examined.
qPCR
The miScript SYBR Green PCR Kit with specific primers (miR-1260b: Ms00020279, miR-720: Ms00014883, miR-576-5p: Ms00007798, miR-1280: Ms00020321, miR-877-5p: Ms00032123, miR-320a: Ms00014707; Qiagen, Hilden, Germany) and the miScript universal primer (Ce-miR-39: Lot. No. 176135137, Qiagen) was used for qPCR-based validation assays, analyzing plasma miRNA levels in samples from ESCC patients (n=96) and healthy controls (n=51).
The ViiA7 real-time PCR System (Applied Biosystems, USA) was used for all qPCR reactions, which were repeated in triplicate with the following thermocycler settings: 95 ℃ for 15 minutes; 40 cycles of 94 ℃ for 15 seconds, 55 ℃ for 30 seconds, and 70 ℃ for 30 seconds; followed by a melting curve (90 ℃ for 10 seconds, 60 ℃ for 30 seconds, and 90 ℃ 10 seconds). Samples were analyzed in triplicate, and relative expression was normalized to spiked-in Ce-miR-39 levels. The 2−ΔΔCt method was used to calculate fold-change values.
Statistical analysis
All miRNA array data analyses were performed using the afore mentioned tool. Statistical testing was conducted using SPSS 17.0 (SPSS Inc., USA), with P<0.05 as the significance threshold. Plasma miRNA profile differences between cohorts were compared via Mann-Whitney U-tests, while correlations between miRNA levels and clinicopathological findings were assessed via Chi-squared test or Fisher's exact probability test. The potential value of specific plasma miRNAs as diagnostic biomarkers of ESCC was assessed based upon the area under curve (AUC) of the receiver-operating characteristic (ROC). Patient survival was measured as the time from surgery until death or the most recent follow-up (November 4, 2015). Survival outcomes were analyzed via the Kaplan-Meier approach. All statistical tests were two-sided.
RESULTS
miR-1260b and miR-720 showed significant differences in ESCC patient plasma relative to healthy controls
The study began by conducting a miRNA microarray analysis comparing miRNA expression profiles in ESCC patients to healthy controls, with Ce-miR-39 as a spiked-in control. Based upon the selected differential expression criteria (fold change>2, P<0.05), 11 miRNAs (miR-205-5p, miR-99b-5p, miR-23b-5p, miR-424-3p, miR-1260b, miR-576-5p, miR-1280, miR-4323, miR-574-3p, miR-877-5p, miR-320a) were upregulated and one (miR-720) was found downregulated significantly in ESCC plasma samples compared with controls (Fig. 1A, 1B, and Table 1).
Figure
1.
Microarray-based identification of miRNAs differentially expressed in ESCC patients and healthy controls.
A: Fold-change values in the expression of miRNAs in ESCC patients relative to healthy controls are shown using a Volcano plot, where red and green points were 2-fold up- or down-regulation respectively. B: Differentially expressed miRNAs in ESCC patients were arranged via hierarchical clustering with green and red respectively corresponding to low and high levels of miRNA expression.
Then six of these miRNAs (miR-720, miR-1260b, miR-576-5p, miR-1280, miR-877-5p, and miR-320a) were selected for qPCR validation in plasma samples from 96 ESCC patients and 51 healthy volunteers. This analysis revealed that the expression profiles of miR-1260b and miR-720 were consistent with the microarray results, with both being significantly differentially regulated in ESCC patients relative to controls (P<0.001 and P=0.001, respectively) (Fig. 2). While miR-1280, miR-877-5p, and miR-320a exhibited comparable differential expression directionality in our qPCR and microarray results, the differences between ESCC patients and controls were not significant in these validation analyses (P>0.05). The miR-576-5p expression was not consistent in qPCR and microarray analyses. As such, miR-1260b and miR-720 were selected for subsequent validation.
Figure
2.
Validation of differential plasma miR-1260b and miR-720 levels in ESCC patients and controls.
The indicated miRNAs were assessed in plasma from ESCC patients (n=96) and controls (n=51) by qPCR, with Ce-miR-39 being used for normalization. Data are means ± SEM, and assays were repeated in triplicate. A: Plasma miR-1260b level was significantly increased in ESCC patients relative to controls (**P<0.001). B: Plasma miR-720 level was significantly decreased in ESCC patients relative to controls (**P<0.001).
The results revealed miR-1260b to be significantly upregulated in ESCC patient plasma relative to healthy control plasma (2.24, 1.41 respectively, P=0.000), whereas the opposite was true for miR-720 (0.66, 2.27 respectively, P=0.001).
miR-1260b and miR-720 were excellent tools capable of discriminating ESCC patients from controls
A ROC analysis revealed that these two miRNAs could reliably identify ESCC patients. Plasma miR-1260b yielded an AUC of 0.695 (P=0.001, Fig. 3A) with respective sensitivity and specificity values of 74.2% and 64.7%, when used to discriminate between ESCC patients and healthy controls. We further found that miR-720 was significantly downregulated in ESCC patients relative to controls (P=0.001, Fig. 2B), yielding an AUC of 0.660 when used as a diagnostic biomarker to discriminate between ESCC patients and controls (P=0.001, Fig. 3B), with respective sensitivity and specificity values of 51.0% and 76.3%. As such, both miR-1260b and miR-720 in isolation represent excellent tools capable of discriminating ESCC patients from controls.
Figure
3.
ROC curves to compare the efficacy of each miRNA, or combination of miRNAs, and to distinguish between ESCC patients and healthy controls.
A: The results of the ROC curve analysis of the miR-1260b assay for detecting ESCC. The analysis revealed that at the optimal cut-off value of 1.144 for miR-1260b, the sensitivity was 74.2% and the specificity was 64.7%, with an AUC of 0.695 (95% confidence interval, 0.606–0.784). B: The results of the ROC curve analysis when using miR-720 to detect ESCC. The ROC analysis revealed that at the optimal cut-off value of 0.267 for miR-720, the sensitivity was 51.0% and the specificity was 76.3%, with an AUC of 0.660 (95% confidence interval, 0.562–0.758). C: The results of the ROC curve analysis when using a combination of miR-1260b and miR-720 ([miR-1260b]/[miR-720]) to detect ESCC. The ROC analysis revealed that at the optimal cut-off value of 0.319 for the (miR-1260b)/(miR-720), the sensitivity was 86.3% and the specificity was 73.2%, with an AUC of 0.814 (95% confidence interval, 0.742–0.882).
Combination plasma miRNA analyses represented a more powerful tool for the diagnosis of ESCC patients
The combined diagnostic efficacy of both miR-1260b and miR-720 as a means of differentiating ESCC and healthy controls was next evaluated, yielding an AUC of 0.814 (P=0.001, Fig. 3C) and sensitivity and specificity values of 86.3% and 73.2%, respectively. This suggests that the plasma levels of these two miRNAs may be reliably evaluated to facilitate the diagnosis of ESCC patients.
The clinical significance of miR-1260b and miR-720 expression in ESCC
The correlation between plasma miR-1260b and miR-720 levels and ESCC patient clinicopathological characteristics was evaluated. For these analyses, 96 ESCC patients were separated into two groups based upon their relative plasma miR-1260b and miR-720 levels as compared to the median levels in this patient cohort (high/low). Chi-squared tests revealed miR-1260b and miR-720 levels to be unrelated to patient age, gender, TNM stage, tumor site, or pathological differentiation. A trend towards a differential miR-1260b expression between patients with stage Ⅰ–Ⅱ and patients with stage Ⅳ was observed, although the difference was not significant (Fig. 4, P=0.067).
Figure
4.
The expression level of miR-1260b in different TNM stages.
Error bars in the figure are represent as Mean ± SEM. Ⅰ: TNM stage Ⅰ; Ⅱ: TNM stage Ⅱ; Ⅲ: TNM stage Ⅲ.
The relationship between plasma miR-1260b and miR-720 levels and ESCC patient prognosis
Kaplan-Meier analyses were used to assess the relationship between the expression of these miRNAs and ESCC patient survival, stratifying patients into two groups based upon whether their plasma miR-1260b and miR-720 levels were above or below the median level (high/low). Subsequent Log-rank analysis indicated that the overall survival of patients expressing high levels of miR-1260b was significantly decreased relative to that of patients expressing low levels of this miRNA (P=0.021, Fig. 5A). In contrast, higher plasma miR-720 levels trended towards being linked to prolonged patient survival, although this relationship was not significant (P=0.109, Fig. 5B).
Figure
5.
The relationship between plasma miR-1260b and miR-720 levels and ESCC patient prognosis.
A: The prognostic value of the miR-1260b expression level. The Kaplan-Meier analysis and Log-rank test showed that there was a significant difference (P=0.021) between patients with higher and lower levels of plasma miR-1260b expression. Overall survival was significantly shorter in the high expression miR-1260b group. B: There were longer survival time in higher expression level than lower expression level of plasma miR-720, but this was not statistically significant (P=0.109).
The ability of miRNAs to regulate cell proliferation, survival, and signaling underscores their frequent dysregulation in a range of tumor types, as demonstrated in prior high-throughput analyses[19-20]. While certain miRNAs that serve as tumor suppressors are often downregulated in tumors, others that serve an oncogenic function may instead be upregulated to facilitate tumor progression[21]. As such, we herein conducted a comprehensive analysis of plasma miRNA profiles in ESCC patients and healthy controls.
The microarray-based analysis led to the identification of 12 miRNAs that were significantly differentially regulated in ESCC patient plasma relative to healthy plasma (11 upregulated, 1 downregulated). Subsequent qPCR-based validation confirmed that miR-1260b was overexpressed in ESCC patients relative to controls, whereas miR-720 was downregulated in ESCC patients.
Using ROC curves, the efficacy of miR-1260b and miR-720 to serve as biomarkers of ESCC status was explored, yielding respective AUC values of 0.695 and 0.660 (both P<0.05). When miR-1260b and miR-720 were combined via a logistic regression approach, the AUC rose to 0.814 and the combined model exhibited excellent sensitivity and specificity as a means of differentiating between ESCC patients and controls (P<0.001, Fig. 3). These results highlight the potential efficacy of miRNAs as ESCC diagnostic biomarkers.
Consistent with our results, one prior group reported that miR-1260b was significantly upregulated in prostate cancer tissue, with such upregulation being associated with more advanced diseases[22]. Furthermore, miR-1260b can promote the proliferation and invasion of lung adenocarcinoma by Wnt/β-catenin signaling pathway[23]. With one report having suggested miR-1260b expression to be specific to infiltrative myxofibrosarcoma[24], and another report identified miR-1260b expression to be linked to non-small cell lung cancer (NSCLC) metastasis[25].
In line with prior reports, miR-1260b expression was elevated in patients with stage Ⅲ of ESCC relative to those with stage Ⅰ or Ⅱ of ESCC, although the difference was not significant (Fig. 4, P=0.067).
While miR-720 has previously been reported to be upregulated in colorectal cancer[26], cervical cancer[27], and glioma[28], it can also serve as a tumor suppressor that is downregulated in breast cancer[29] and ESCC[30]. Noteworthy, Shinozuka reported that the level of miR-720 was lower expression in ESCC tumor tissues compared to adjacent normal tissues[30]. In fact, this observation supports the current study's data. Consistent with these prior reports, we found miR-720 to be significantly downregulated in ESCC patient plasma.
The study additionally observed significant correlations between miRNA expression and ESCC patient survival, with miR-1260b being expressed at significantly higher level in ESCC patients relative to controls, and associated with shorter overall ESCC patient survival. This study suggests that miR-1260b may play role in the progression of ESCC and may be a prognostic factor of ESCC.
In summary, the results indicated that plasma miR-1260b and miR-720 levels were promising diagnostic and prognostic biomarkers for use in the evaluation of ESCC patients. In particular, there was higher diagnostic performance to distinguish between ESCC and healthy controls when miR-1260b and miR-720 were combined. Meanwhile, high level of miR-1260b was associated with a bad prognosis of ESCC so that miR-1260b may be a prognosis factor of ESCC. These results will help to further study the mechanism of ESCC progression. In addition, plasma miRNA can be developed as a useful diagnostic approach with sensitive, specific, and non-invasive diagnosis of early-stage ESCC.
Acknowledgments
This research was supported by grants from the National Nature Science Foundation of China (Grant No. 81972840), Key Research and Development Project of Shanxi Province (Grant No. 201903D321129) and Medical Key Science Project of Shanxi Province (Grant No. 2020XM52).
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