File: cbm480.tex; BOKCTP/wyn p. 1
Cancer Biomarkers 00 (2015) 1–9 DOI 10.3233/CBM-150480 IOS Press
Serum microRNA-218 is a potential biomarker for esophageal cancer Zhaojing Jianga,1 , Qiancheng Songb,1 , Suoping Yanga,1, Rong Zenga, Xufeng Lia , Chunyu Jianga,c, Weimin Dinga , Jiren Zhanga and Yanfang Zhenga,∗ a
Oncology Center, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong, China Institute of Genetic Engineering, Southern Medical University, Guangzhou, Guangdong, China c Department of Oncology, Anyang Cancer Hospital, Anyang, Henan, China
Abstract. BACKGROUND: Several studies demonstrated that microRNAs are stably detectable in plasma/serum and are potential biomarkers for some diseases. The expression of microRNA-218 (miR-218) is downregulated in esophageal cancer as reported in previous research. OBJECTIVE: The purpose of this study is to investigate whether miR-218 can be served as a serum biomarker for esophageal cancer. METHODS: We tested the expression level of miR-218 in serum of 106 patients with esophageal cancer and 60 healthy volunteers by RT-PCR and analyzed the relationship between serum miR-218 expression and the clinical characteristics. RESULTS: The serum expression of miR-218 was significantly lower in patients with esophageal cancer than that in healthy individuals. The value of the area under the receiver-operating characteristic (ROC) curve (AUC) was 0.833. Furthermore, the ROC curves to detect early esophageal cancer with Tis-T1 or Stage 0-I showed AUCs of 0.825 and 0.829, respectively. In the esophageal cancer group, the serum expression of miR-218 was found to be lower in esophageal cancer patients with poorer differentiation, later stage, and lymph node metastasis, highlighting that low serum expression of miR-218 may be related to tumor development and progression in esophageal cancer. CONCLUSIONS: The serum expression of miR-218 is downregulated in esophageal cancer patients and is correlated with tumor differentiation, stage, and lymph node metastasis. Serum miR-218 may be a potential biomarker for early detection and clinical evaluation in patient with esophageal cancer.
Keywords: Esophageal cancer, microRNA, microRNA-218, serum biomarker, cancer detection
Esophageal cancer (EC) is the sixth leading cause of cancer death worldwide and the eighth most common type of cancer worldwide . There is 3 to 4 times more esophageal cancers among males than females. Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are the two
3 4 5 6 7
authors contributed equally to this work and are considered to be co-first authors. ∗ Corresponding author: Yanfang Zheng, Oncology Center, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, Guangdong, China. E-mail: [email protected]
main histological types, according to different etiologic and pathologic characteristics. EAC is common in western countries while ESCC is frequent in the developing world include East Asia and the Caspian belt (also known as the “Central Asian Esophageal Cancer Belt”), especially in China whose incidence in the high-risk northern and central China exceeds 100 cases per 100,000 people per year [2,3]. Approximately 70% of global esophageal cancer cases occur in China, with ESCC as the histopathological form in the vast majority of cases (> 90%) . The survival and prognosis of EC patients depend on the stage of the tumor at the time of detection. Despite significant investment and advances in the treat-
c 2015 – IOS Press and the authors. All rights reserved ISSN 1574-0153/15/$35.00
8 9 10 11 12 13 14 15 16 17 18 19 20 21
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
2. Materials and methods
2.1. Patients and blood samples
From February 2011 to July 2012, a total of 106 patients with esophageal cancer at the Anyang Cancer Hospital (Henan Province, China) were recruited. All patients were pathologically diagnosed as esophageal cancer using surgical specimens and biopsies. Clinicopathological characteristics of all patients were presented in Table 1, and tumor stage and differentiation level was determined using the 2010 AJCC pulished TNM staging classification system . Sixty healthy volunteers matched with age and gender of the esophageal cancer patients were used as control. The control group was defined as healthy individuals
cancer . Takeshita N et al. reported that the miR1246 expression level in the serum from ESCC patients was significantly higher than that from healthy controls. MiR-1246 expression level is negatively associated with 2-year survival . The research conducted by Hirajima S et al. showed that plasma concentrations of miR-18a were significantly higher in ESCC patients than healthy volunteers. Compared with preoperation, Plasma levels of miR-18a were significantly decreased after operation . These circulating microRNAs are all differently expressed in tumor and non-tumor tissues, and associated with clinical characteristics and survival. These findings suggest that serum levels of miRNA can be used as biomarkers in cancer detection and treatment. microRNA (miR)-218 is reported as a tumor suppressor miRNA and is downregulated in many kinds of cancers, such as gastric cancer , head and neck squamous cell carcinoma , glioblastoma , lung cancer , and oral cancer . Downregulated miR218 is correlated with poor prognosis in gastric cancer and restored expression of miR-218 can reduce tumor growth and invasion, and enhance apoptosis . Recently, it has been reported that miR-218 is highly downregulated in human esophageal cancer compared with normal esophageal tissues [29,30]. It has not been reported whether the expression of miR-218 is deregulated in the serum of esophageal cancer patients. Therefore, we investigated the expression of miR-218 in the serum of esophageal cancer patients compared with normal individuals, and analyzed the relationship between miR-218 levels in serum and clinicopathological characteristics.
ment of cancer, the overall survival for advanced and metastatic esophageal cancer is still poor, with a 5year survival rate of less than 15% after surgery in China . In order to improve the prognosis of patients with esophageal cancer, primary tumors must be detected at an early stage, and recurrent disease must be diagnosed when it is still minimal. At present, gastrointestinal endoscopy remains the primary screening tool, by which the suspected lesions can be biopsied for histopathological analysis. Even though it is proved to increase the detection of early tumor and therefore can prolong the survival of the patient, this invasive test is generally considered to be inconvenient and painful. Because of this limitation, it is urgent to find novel predictive markers for EC. Serum and plasma are easily accessible and less invasive, so circulating biomarkers are one of the most promising means of diagnosis. Conventional serum tumor markers, such as carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC), have been used as convenient diagnostic assays for early detection and monitoring the tumor dynamics of esophageal cancer [5,6]. However, these serum tumor markers lack sufficient sensitivity and specificity to facilitate early detection of cancer . Therefore, it is important to detect novel circulating biomarkers using a less invasive and more easily diagnostic assay for esophageal cancer. MicroRNAs (miRNAs) are noncoding, 18–24 nucleotide long, single-stranded RNAs that can negatively regulate the expression of genes involved in many cellular processes, including cell proliferation, apoptosis, migration, invasion, and stress response [8– 12]. It has been shown that deregulated miRNA expression are present in many human carcinomas , and are associated with the pathogenesis, progression, and natural history of several cancers [8,14]. Tumorassociated miRNAs are previously reported to exist in serum and plasma of cancer patients [15,16]. Although the mechanism underlying their secretion is still being elucidated, the miRNAs present in the blood stream (circulating miRNAs) have been shown to be remarkably stable, even in the RNase-rich environment of the blood. Furthermore, the miRNA levels were found to be independent of the subject age and sex, and are similar in plasma and serum [17,18], which hold great promise as a new class of biomarkers for cancer detection and prognosis [19–22]. The presence of circulating microRNAs have been reported in diffuse large B-cell lymphoma , non-small cell lung cancer , nasopharyngeal carcinoma , and prostate
Z. Jiang et al. / Serum microRNA-218 is a potential biomarker for esophageal cancer
File: cbm480.tex; BOKCTP/wyn p. 2
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
109 110 111 112 113 114 115 116 117 118 119
File: cbm480.tex; BOKCTP/wyn p. 3
Z. Jiang et al. / Serum microRNA-218 is a potential biomarker for esophageal cancer
Table 1 The association between the serum miR-218 expression and clinicopathologic parameters of esophageal cancer patients miR-218 expressiona (Median ± SD)
0.4940 ± 0.4401 0.5236 ± 0.3971
0.4781 ± 0.3880 0.5661 ± 0.4677
0.3878 ± 0.2651 0.5481 ± 0.4505
19 68 19
0.4969 ± 0.4283 0.5432 ± 0.4436 0.3974 ± 0.2880
0.5250 ± 0.4302 0.3530 ± 0.2219
12 70 24
0.7871 ± 0.4236 0.5328 ± 0.4182 0.2997 ± 0.3117
Number of subjects (N = 106)
0.5471 ± 0.3681 0.4993 ± 0.4302
20 48 38
Characteristics Age (years) 62b > 62 Gender Male Female Family history of EC Yes No Tumor location Upper esophagus Middle esophagus Low esophagus Histology ESCC EAC Differentiationc Well Moderate Poor T stagec Tis-1 T2-3 Staged 0-I II III Lymph node status Negative Positive
0.5510 ± 0.3511 0.6460 ± 0.4689 0.3133 ± 0.2955
0.6389 ± 0.4421 0.3255 ± 0.2991
serum miR-218 expression is normalized to the serum miR-16 expression; b The median age of all esophageal cancer patients in this study; stage and differentiation level was determined using the 2010 AJCC published TNM staging classification system; d TNM stage; P < 0.05, is considered as statistically significant.
2.2. RNA isolation and qRT-PCR
From serum samples, total RNA was isolated with the mirVana PARIS kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Briefly, 400 μl of serum were incubated with an equal volume of Denaturation Solution for 5 min on ice. RNA extraction was performed by the acid–phenol: chloroform method, and precipitation was carried out with ethanol and a filter cartridge. The extracted RNA was eluted in 100 μl of preheated Elution Solution. The RNA concentration and purity were determined us-
122 123 124 125 126
130 131 132 133 134 135 136 137 138
who visited hospital for routine checkup, and they did not have any esophageal lesions or a history of malignancy. Permission was obtained from the hospital Ethical Committee, and written informed consent was provided by all participates. Two milliliter venous blood samples were obtained from each participate before any treatment and immediately centrifuged; sera and other components were stored at −80◦C.
ing NanoDrop 2000 Spectrophotometer (Thermo Scientific). The RNA samples were immediately stored at −80◦C until use. Reverse transcription reactions and Real-time PCR was performed with Hairpin-itTM miRNAs RT-PCR Quantitation Kit (GenePharma, Shanhai, China). The reverse transcription reaction was carried out in 20 μl containing 4 μl of 5 × MMLV RT Buffer, 0.75 μl of 10 mM dNTP, 1.2 μl of miR-218 RT primers (1 μM), 1.2 μl of U6 RT primers (1 μM) or 1.2 μl of miR-16 RT primers (1 μM), 0.2 μl of MMLV Reverse Transcriptase (200 Ul/1) and 1 μg RNA extract. For the synthesis of cDNA, reaction mixtures were incubated at 25◦ C for 30 min, at 42◦ C for 30 min, and at 85◦ C for 5 min and then held at 4◦ C. Next, 2 μl of cDNA solution was amplified using 10 μl of 2 × Real-time PCR Master Mix, 0.32 μl of miRNAs specific Primer set (5 μM), 0.2 μl of Taq DNA polymerase (5 Ul/1) and 7.48 μl of ddH2 O in a final volume of 20 μl. Quantitative PCR was run on a ABI PRISM 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA) and re-
139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159
165 166 167 168 169
2.3. Statistical analysis
Statistical analyses were performed with SPSS 13.0 software (SPSS, Inc, Chicago, IL). The Mann-Whitney U-test was performed to compare the differences in the serum miRNA expression levels between the cancer patients and the healthy control volunteers. The Mann-Whitney U-test and the Kruskal-Wallis test were used to evaluate the correlations between the results of the serum miRNA expression levels and the clinicopathological factors. Receiver-operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to assess the feasibility of using the serum miRNA expression levels as diagnostic markers for detecting esophageal cancer. The Younden Index was used to determine the cutoff value for plasma miRNA concentrations . All tests were two-sided, and the significance level was set at p < 0.05. Data obtained from qRT-PCR were expressed as median ± standard deviation (SD).
176 177 178 179 180 181 182 183 184 185 186 187 188
3.1. Serum expression of miR-218 in esophageal cancer patients is lower than that in healthy volunteers
193 194 195 196 197 198 199 200 201 202 203 204
Quantitative RT-PCR analysis was performed to demonstrate the differences of serum miR-218 expression in esophageal cancer and healthy individuals. Expression of miR-218 was detectable in serum from 106 esophageal cancer patients and 60 healthy volunteers. The miR-218 expression level in the serum from esophageal cancer patients was significantly lower than that from controls (0.5088 ± 0.4174 vs 1.0028 ± 0.3368, p < 0.001, Fig. 1A) after normalized to serum miR-16 expression. To detect cutoff points that could discriminate the serum miR-218 levels between esophageal cancer patients and healthy volun-
207 208 209 210 211
In order to clarify whether the serum expression level of miR-218 can predict the diagnosis of early esophageal cancer, we compared expression of miR218 in serum between early esophageal cancer patients (Tis-T1 or Stage 0-I) and healthy volunteers. The serum expression of miR-218 of early esophageal cancer patients (Tis-T1 or Stage 0-I) was significantly lower than that of healthy volunteers (0.5471 ± 0.3681vs 1.0028 ± 0.3368, p < 0.001, Fig. 2A; 0.5510 ± 0.3511 vs 1.0028 ± 0.3368, p < 0.001, Fig. 2C;). The values of AUC used to detect early esophageal cancer (Tis-T1 or Stage 0-I) was 0.825 (95% confidence interval, 0.723–0.927; Fig. 2B), 0.829 (95% confidence interval, 0.725–0.933; Fig. 2D), repectively. The diagnostic sensitivity and specificity of early esophageal cancer with Tis-T1 or Stage 0-I at the optimal cutoff value of 0.6831 using ROC curves were 66.7% and 85% (Fig. 3B) or 70% and 85% (Fig. 3D) respectively. Indeed, the sensitivity of serum miR218 for early esophageal cancer detection was much higher than that conventional tumor markers such as SCC, CEA . Our results provided evidence that serum miR-218 level can be used to distinguish early esophageal cancer patients from healthy volunteers with clinically satisfactory sensitivity and specificity.
3.2. Serum expression of miR-218 in early esophageal cancer patients with Tis-T1 or Stage 0-I is lower than that in healthy volunteers
teers, we used ROC curves with the Younden index. The sensitivity was 71.7% and the specificity was 76.7%, with an AUC of 0.833 (95% confidence interval, 0.772–0.893; Fig. 1B) at the optimal cutoff point of 0.7704885 for miR-218. These results indicate that serum miR-218 may be a potential biomarker for esophageal cancer.
action mixtures were incubated at 95◦ C for 3 min, followed by 40 cycles of 95◦ C for 12 sec, and 60◦ C for 40 sec. The primers of the microRNAs in this study are synthesized by GenePharma company which are not provided in the instruction. For data analysis, the expression levels of miRNAs in serum samples were normalized to miR-16 [15,32]. Each assay was performed in triplicate. The relative expression of miR-218 for esophageal cancer and normal controls was calculated by the 2−ΔΔCT method.
Z. Jiang et al. / Serum microRNA-218 is a potential biomarker for esophageal cancer
File: cbm480.tex; BOKCTP/wyn p. 4
3.3. miR-218 expression is associated with clinical characteristics of esophageal cancer To better clarify how serum miR-218 can serve as a potential biomarker for esophageal cancer, the relationship between the serum expression of miR-218 and clinicopathological features in 106 esophageal cancer patients was analyzed (Table 1). There was significant lower serum miR-218 expression in patients with poor differentiation than that in patients with well differentiation and moderate differentiation (0.2997 ± 0.3117 vs 0.7871 ± 0.4236, p = 0.001; 0.2997 ± 0.3117 vs 0.5328 ± 0.4182, p