A five-microRNA signature identified from genome-wide serum microRNA expression profiling serves as a fingerprint for gastric cancer diagnosis

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Abstract

Background

Prognosis of patients with gastric cancer (GC) is generally poor due to the lack of non-invasive tools for GC detection. The purpose of present study was to identify a serum microRNA (miRNA) expression profile that can serve as a novel diagnostic biomarker for GC detection and to assess its clinical applications in monitoring disease progression.

Methods

Serum samples were taken from 164 GC patients and 127 age- and gender-matched tumour-free controls. An initial screening of miRNA expression by Solexa sequencing was performed using serum samples pooled from 20 patients and 20 controls, respectively. Differential expression was validated using hydrolysis probe-based stem-loop quantitative reverse transcription polymerase chain reaction (qRT-PCR) in individuals samples, the samples were arranged in two phases.

Results

The Solexa sequencing results demonstrated that 19 serum miRNAs were markedly upregulated in the GC patients compared to the controls. The qRT-PCR analysis further identified a profile of five serum miRNAs (miR-1, miR-20a, miR-27a, miR-34 and miR-423-5p) as a biomarker for GC detection. The analysis results showed that the expression level of five serum miRNAs was correlated to tumour stage. The areas under the receiver operating characteristic (ROC) curve of this five-serum miRNA signature were 0.879 (95% confidence interval (CI) 0.822–0.936) and 0.831 (95% CI 0.767–0.898) for the two sets of serum samples, respectively, markedly higher than those of the biomarkers carcinoembryonic antigen (CEA) (0.503) and carbohydrate antigen 19-9 (CA19-9) (0.600).

Conclusions

We identified five-miRNA signature for GC diagnosis by genome-wide serum miRNA expression profiling. Expression levels of this serum miRNA-based biomarker also indicate tumour progression stages.

Introduction

Gastric cancer (GC) is the fourth most common human malignant disease and the second most frequent cause of cancer-related death worldwide.1 Currently, surgical resection is the most effective treatment and prolongs the survival of patients with early gastric cancer; however, for advanced gastric cancer, it frequently recurs as nodal and haematogenous metastases and peritoneal dissemination, and the prognosis for individuals with advanced disease remains poor.2 Therefore, improvement in diagnosis and treatment could increase the long-term survival of patients with resectable-stage GCs. Unfortunately, most early-stage GCs are asymptomatic and difficult to detect.3

Chromoendoscopy and random biopsies can diagnose some early-stage GC patients, but the invasiveness of these diagnostic procedures and potential sampling errors with random endoscopic biopsy limit their efficacy. Meanwhile, for the diagnosis of GC, few highly sensitive or highly specific tumour markers are available, and current diagnostic tools for GC, including the serological markers carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA), have low specificity and sensitivity. Several groups have undertaken high-throughput analyses of GC expression profiles by DNA microarrays and microdissection.4 Good markers for diagnosis and progression of GC, however, have not yet been identified. These facts partially account for the extremely poor prognosis and high mortality rate of GC. Therefore, novel biomarkers and diagnostic methods for early detection of GC are urgently needed to reduce the disease morbidity and mortality.

MicroRNAs (miRNAs) are a subset of non-coding RNA molecules (21–23 nucleotides in length) that are believed to regulate gene expression.5 Altered expression of miRNAs has been associated with several diseases, particularly cancer.6 Using tissue miRNA expression profiles as prognostic biomarkers in cancer has been demonstrated by several studies.7, 8, 9, 10 Because serum and plasma are relatively easy to access, circulating biomarkers are one of the most promising means of diagnosis. Previous studies from our group and others have shown that human serum contains miRNAs, and that the expression pattern of these serum miRNAs can potentially be used to identify various types of cancer, including prostate cancer, large B-cell lymphoma, ovarian cancer, liver cancer and non-small cell lung cancer.11, 12, 13, 14, 15, 16 Therefore, identifying a unique serum miRNA expression profile in GC can potentially assist tumour diagnosis and cancer treatment.

To ascertain whether a serum miRNA expression signature can distinguish GC from cancer-free controls, we conducted genome-wide serum miRNA expression profiling by Solexa sequencing followed by a stem-loop quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay using hydrolysis probes to systematically and extensively evaluate serum miRNA expression. By statistical analysis, we obtained a profile of five serum miRNAs, which can serve as a biomarker for GC detection. The correlation between serum miRNAs and GC progression was further assessed.

Section snippets

Study design, patients and control subjects

A multi-stage, case–control study was designed to identify a serum miRNA profile as a surrogate marker for GC (Fig. 1). All samples were collected from consenting individuals according to the protocols approved by the ethics committee of each participating institution. In total, 164 patients with primary GCs and 127 control subjects were enrolled in our study. In the initial biomarker screening stage, GC serum samples pooled from 20 non-metastatic GC and 20 metastatic GC patients and control

Description and clinical features of the patients

All 164 patients enrolled in the present study were clinically and pathologically diagnosed with GC. As shown in Table 1, there was no significant difference in the distribution of smoking (P = 0.346), alcohol consumption (P = 0.214), age (P = 0.858) and gender (P = 0.232) between the cancer patients and the normal subjects. Among the 164 patients, 29 (18%), 56 (34%), 48 (29%) and 23 (14%) were classified as stages I, II, III and IV, respectively. Elevated levels of CEA (>5 U/ml) and CA19-9 (>37 U/ml)

Discussion

Dysregulation of miRNA expression in cancer has been widely reported previously.24 However, the studies mainly focused on miRNAs expressed in tumour tissues and cells, Katada and colleagues25 demonstrated that the survival rate was significantly lower in undifferentiated GC patients with high expression levels of miR-20b or miR-150 and that miR-27a expression was correlated with lymph node metastasis. Chan and colleagues26 found that miR-21 was overexpressed in 92% of the GC patients. Although

Role of the funding source

Test materials, equipment, testing, etc. are supported by the fund.

Conflict of interest statement

None declared.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (30890044, 30772484, 30971344 and 30570731), Key Science Project supported by the Government of TianJin (10JCZDJC18400).

References (34)

  • N. Yang et al.

    MicroRNA microarray identifies Let-7i as a novel biomarker and therapeutic target in human epithelial ovarian cancer

    Cancer Res

    (2008)
  • M.D. Mattie et al.

    Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies

    Mol Cancer

    (2006)
  • X. Chen et al.

    Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases

    Cell Res

    (2008)
  • P.S. Mitchell et al.

    Circulating microRNAs as stable blood-based markers for cancer detection

    Proc Natl Acad Sci USA

    (2008)
  • S. Gilad et al.

    Serum microRNAs are promising novel biomarkers

    PLoS One

    (2008)
  • K. Wang et al.

    Circulating microRNAs, potential biomarkers for drug-induced liver injury

    Proc Natl Acad Sci USA

    (2009)
  • E.K. Ng et al.

    Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening

    Gut

    (2009)
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    These authors contributed equally to this work.

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