Introduction

Gastric cancer is the second leading cause of cancer death in the world and will likely remain as one of the leading causes of all deaths in the near future [1, 2]. However, few specific tumor markers with high sensitivity and high specificity are available for the diagnosis of gastric cancer. In addition, the sensitivity of the existing serum biomarkers, carcinoembryonic antigen (CEA), CA19-9, or CA72-4, is only about 40% [3–6].

Small noncoding microRNAs (miRNAs) have been shown to play crucial roles in diverse biological processes, such as cell development, signal transduction, tissue differentiation and maintenance, disease, and carcinogenesis [7, 8]. miRNAs can contribute to cancer development and progression, and are differentially expressed in normal tissues and cancers [9]. These observations suggest that miRNAs may potentially act as tumor suppressor genes or oncogenes, respectively [10].

The development and metastasis of gastric cancer are characterized by multiple genetic alterations. Previous studies showed altered expression levels of several miRNAs in gastric cancer [9, 11–13]. Volinia et al. found that 26 miRNAs that were over-expressed and 17 miRNAs that were down-regulated in six kinds of cancers including stomach [9]. miR-21 was found to be over-expressed in 92% (34/37) of gastric cancer tissues, and may serve as an efficient diagnostic marker for gastric cancer [11]. The expression of let-7a was found to be reduced in gastric tumors compared to that in normal gastric tissue [12]. A recent study showed that the restoration of miR-34 expression re-establishes the tumor-suppressing signaling pathway in human gastric cancer cells lacking functional p53 [13]. Therefore, miR-34 is involved in the p53 tumor suppressor network.

Recently, we used microarray technology to conduct the genome-wide expression profiling of miRNAs in gastric cancer, and found 19 differentially expressed miRNAs in gastric cancer tissue when compared to their expression in non-tumor tissues [14]. Among the 19 miRNAs, miRNA-31 (miR-31) was expressed at the lowest levels in gastric cancer. A link between miR-31 expression and gastric cancer has not been established. Moreover, the role of miR-31 in gastric cancer remains to be investigated. As a result, the objective of the present study was to elucidate the value of miR-31 in the diagnosis of gastric cancer.

Materials and methods

Patients and specimens

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue samples were obtained from surgical specimens from 63 patients (33 male, 30 female; 66.06 ± 5.38 and 51.73 ± 7.65 years old, respectively) diagnosed with gastric cancer, from May 2005 to December 2007 at Ningbo No. 2 Hospital, China. Non-tumor gastric tissues were randomly selected from 10 of these patients (7 male, 3 female; 57.86 ± 6.57 and 52.33 ± 9.61 years old, respectively) and used as controls. Informed consent was obtained from all subjects, and the Human Research Ethics Committee from Ningbo University approved all aspects of the study. Tumors were staged using the tumor–node–metastasis (TNM) staging of the International Union Against Cancer [15]. Histological grade was assessed according to the World Health Organization criteria [16]. The non-tumor tissues were more than 1.5 cm from the tumor and were confirmed as such by an experienced pathologist. Another five non-tumor tissues samples that were more than 3 cm from the gastric cancer were also detected. Similar non-tumor tissues samples from patients with colon, lung, and breast cancers were also assayed, respectively. Two samples were obtained for each cancer type.

Total RNA preparation

Total RNA from human FFPE tissues was isolated using the RecoverAllTM Total Nucleic Acid Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. The concentration and purity of the total RNA samples were assessed using the SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA, USA).

Reverse transcription

The cDNAs were generated using the miScript Reverse Transcription (RT) Kit (Qiagen GmbH, Hilden, Germany). According to the manufacturer’s instructions, 1 μg total RNA, 1 μl miScript Reverse Transcriptase Mix, and 4 μl miScript RT buffer were mixed well and incubated for 60 min at 37°C. All reverse transcriptions and no-template controls were run at the same time.

Real-time PCR for detection of miR-31

Real-time polymerase chain reaction (PCR) was performed using the miScript SYBR Green PCR Kit (Qiagen) on an Mx3005P QPCR System (Stratagene, La Jolla, CA, USA). The 20 μl PCR mixture included 2 μl reverse transcription product, 10 μl 2× QuantiTect SYBR Green PCR Master Mix, 2 μl 10× miScript Universal Primer, 2 μl 10× miScript Primer Assay (for miR-31; Qiagen), and 4 μl autoclaved distilled water. The reaction mixtures were incubated at 95°C for 10 min, followed by 40 amplification cycles of 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s. The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence passed the fixed threshold. We also quantified transcripts of U6 small RNA using the Hs_RNU6B_2 miScript Primer Assay (Qiagen) to normalize the levels of miR-31.

The ΔCt method was used for analysis [17]. First, the cycle number at the threshold level of fluorescence (Ct) for each sample was determined. Next, the ΔCt value was calculated. The ΔCt value was the difference between the Ct value of miR-31 and the Ct value of U6: ΔCt = Ct (miR-31) − Ct (U6). Finally, the ΔΔCt value and the normalized miR-31 expression were calculated: ΔΔCt = ΔCt (cancer tissues) − ΔCt (non-tumor tissues). Normalized miR-31 expression in a sample = 2−ΔΔCt. The experiment was repeated thrice. The investigators were blinded to the results of clinical and pathological diagnoses.

Detection of serum CEA level

For determination of CEA, serum samples (25 μl, undiluted) were measured using the fully automatic, competitive chemiluminescent immunoassay as reported previously [18]. The cutoff value recommended for diagnostic purposes was 10 μg/l [18]. A concentration value above the cutoff value was considered positive.

Statistical analysis

Statistical analysis was performed using Statistical Program for Social Sciences (SPSS) software 13.0 (SPSS Inc., Chicago, IL, USA). Clinicopathological factors and miR-31 levels were analyzed by one-way analysis of variance (ANOVA). The level of significance was set at P < 0.05.

Results and discussion

Expression of miR-31 was down-regulated in gastric cancer

By real-time RT-PCR, the relative miR-31 expression levels were measured. Then, the expression levels of miR-31 in cancer tissues from patients with gastric cancer were compared with those in non-tumor tissues. We found that miR-31 expression was down-regulated in the patients (P: 0.044, Fig. 1), with an average 6.28-fold decrease.

Fig. 1
figure 1

Level of miR-31 expression in gastric cancer tissues (n = 63) was lower than that in non-tumor tissues (n = 10). Values shown (ΔCt) are relative to those of U6 small RNA

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Expression levels of miR-31 in different types of tissues were variable

To confirm our results, we assayed additional tissues from a distance greater than 1.5 cm from the gastric cancer (3 cm from the tumor) and from non-cancerous different tissue. We found that the expression levels of miR-31 in gastric cancer tissues were lower than those in non-cancerous gastric tissues (3 cm from the tumor), colon tissues, and lung tissues, while higher than those from breast tissues (Fig. 2).

Fig. 2
figure 2

Expression levels of miR-31 in different types of tissues were variable. Values shown (ΔCt) are relative to those of U6 small RNA. Low ΔCt indicates low expression

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Relationship of miR-31 expression levels in cancer tissues and clinicopathological factors in patients with gastric cancer

We investigated the relationship between miR-31 expression levels in cancer tissues and clinicopathological factors in patients with gastric cancer. We found that the expression level of miR-31 was not associated with the TNM stage (P: 0.399, Table 1). As shown in Table 1, miR-31 expression levels were not associated significantly with tumor size and differentiation, lymphatic and distant metastasis, and invasion.

Table 1 Relationship of miR-31 expression levels in cancer tissues with clinicopathological factors in patients with gastric cancer
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Detection of miR-31 is better than detection of serum CEA for the diagnosis of gastric cancer

Serum CEA is the most commonly used tumor marker for the diagnosis of gastric cancer. Therefore, we compared the detection rate of CEA with that of with miR-31. We used a cutoff value of 0.4, with samples having a ΔCt value < 0.4 considered positive for down-regulation [19]. As shown in Table 2, the miR-31-positive detection rate can reach 68.29%. However, the serum CEA-positive detection rate is only 21.95%. Considering our observation that miR-31 expression levels are not associated with the clinicopathological factors of patients with gastric cancer, we suggest that miR-31 may be an efficient diagnostic marker for gastric cancer, which does not affect the clinical prognosis of gastric cancer patients.

Table 2 Comparison of the miR-31-positive detection rate with the serum CEA-positive detection rate
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Although the number of verified human miRNAs is expanding, only a few have been described functionally. However, emerging evidence suggests the potential involvement of the altered regulation of miRNA expression in the pathogenesis of cancers and that these genes are thought to function as both tumors suppressor and oncogenes.

The miR-31 gene is located at 9p21.3. It is abnormally expressed in cancer cells and may play an important role in cancer development. Bandrés et al. [10] found that miR-31 was one of the most significantly deregulated miRNAs in colorectal cancer tissues. They also observed that the expression level of miR-31 is correlated with the stage of this type of cancer. Yan et al. [20] used a microarray containing 435 mature human miRNA oligonucleotide probes to investigate the global expression profile of miRNAs in primary breast cancer and normal adjacent tumor tissues, and found that miR-31 and 6 others miRNAs were down-regulated greater than two-fold. Shen et al. used ischemia-induced retinal neovascularization as a model to investigate the possible role of miRNAs in a clinically important disease process. Microarray analysis and real-time RT-PCR demonstrated that miR-31 expression was substantially decreased in the ischemic retina. In addition, the injection or enhanced expression of miR-31 reduced ischemia-induced retinal neovascularization significantly [21]. Another study used granulocytes isolated from patients with primary myelofibrosis to investigate the expression profiling of miRNAs [22]. The results showed that in primary myelofibrosis granulocytes, the level of miR-31 was significantly lower than that in normal controls and patients with polycythemia vera or essential thrombocythemia.

Interestingly, other studies found that miR-31 expression was up-regulated in cancer cells. Array-based miRNA profiling was performed on hepatocellular carcinoma cells that were derived from chronic carriers of hepatitis B virus and hepatitis C virus, and nonviral-associated patients [23]. The results showed that distinct up-regulation of miR-31 expression. Wang et al. [24] reported that the expression levels of 156 human mature miRNAs were examined using real-time quantitative PCR on laser microdissected cells of four tongue carcinomas and paired normal tissues. miR-31 expression was found to be up-regulated at least three-fold.

In conclusion, the expression levels of miR-31 are different in different tumor types. miR-31 may be a useful as tumor marker in the diagnosis of gastric cancer and other cancers.