A total of 68 patients with pathologically diagnosed gastric cancer were recruited from January 2009 to June 2010 at the First Affiliated Hospital of Henan University of Science and Technology (Henan Province, China). Blood samples were obtained before any treatment and immediately centrifuged; sera and other components were stored at -80 °C. Blood samples from 56 healthy people were used as controls. The control group was defined as healthy individuals who visited hospital for routine check-up, and they did not have any gastric lesions or a history of malignancy. Permission was obtained from the hospital Ethical Committee, and written informed consent was provided by all patients.

TRIzol reagent (CWbio Co. Ltd., Beijing, China) was used to isolate total RNA from the snap-frozen tissues. The isolated RNA was treated with DNase I (Invitrogen, Carlsbad, CA, United States). The RNA concentration and purity were determined using NanoDrop ND-1000. The ratio of 28S/18S was analyzed by Glyko Bandscan 5.0. RNA quality and quantity were determined by spectrophotometer (Wilmington, DE, United States) at 260 and 280 nm. Reverse transcription of RNA was performed using the NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen, United States). qRT-PCR was performed with the Light Cycler 2.0 Real-Time PCR System (Roche, Germany) in a total volume of 20 μL in glass capillaries containing 2 μL cDNA, 0.8 μL each primer, and 10 μL Light Cycler TaqMan Master Mix (Invitrogen). The PCR for the miR-21 gene was initiated using a 10-min denaturation step at 95 °C followed by termination with a 30-s cooling step at 40 °C. The cycling protocol consisted of denaturation at 95 °C for 15 s and annealing at 60 °C for 60 s; this cycle was performed 40 times. Fluorescence detection was performed at the end of each extension step. The PCR products were confirmed by melting curve analysis. For data analysis, we used the cel-miR-39 as an endogenous control. All procedures were repeated three times. The relative expression of miR-218 for gastric cancer and normal controls was calculated by the 2^-ΔΔCT method. The mean relative expression of miR-218 in the serum of healthy people was set as N, and all relative expressions of miR-218 in samples of gastric cancer were compared with it. Based on the ratios, we determined low miR-218 expression (T/N < 1.2); medium expression (T/N = 1.2-10); and high expression (T/N > 10).

Differences of miR-218 expression in the two groups were assessed by one-way analysis of variance. The Mann-Whitney U test was used to determine the associations of miR-218 expression and Gastric Cancer clinicopathological features. Survival functions and differences were calculated by the Kaplan-Meier method and assessed using the log-rank statistic. Multivariable survival analyses were performed using the Cox proportional hazards regression model. SPSS version 17.0 was used for statistical analysis. Statistically significant level was defined as P < 0.05.

Demographic and clinicopathological variables for the cohort are summarized in Table 1. Sixty-eight patients with gastric cancer were included in this study (46 male and 22 female) aged 35-82 years (mean = 63 years). Pathological classification of tumor grading resulted in 18 poorly differentiated, 24 moderately differentiated and 26 well differentiated tumors, and T staging resulted in 19 as T1, 17 as T2, 15 as T3, and 17 as T4. There were 28 cases of metastatic gastric cancer and 40 non-metastatic cases. Follow-up for the entire cohort ranged from 18 to 36 mo (mean = 29.6 mo).

qRT-PCR analysis was performed to demonstrate the differences in miR-218 expression in gastric cancer and normal serum. Expression of miR-218 was significantly decreased in gastric cancer compared with normal serum (1.15 ± 0.08 vs 0.37 ± 0.023, P = 0.026, Figure 1A). Furthermore, compared with the earlier stages (stage I and II) and non-metastasis, the later stages (stage III and IV) (0.48 ± 0.023 vs 0.22 ± 0.011, P = 0.023 and 0.45 ± 0.021 vs 0.19 ± 0.011, P = 0.031; Figure 1B and D) and metastasis had lower serum expression of miR-218. There was a significant difference between the well-differentiated and poorly differentiated tumors in serum expression of miR-218 (0.49 ± 0.021 vs 0.21 ± 0.019, P = 0.025, Figure 1C). Moreover, there was a significant decrease in miR-218 expression in the serum of gastric cancer patients compared to the healthy individuals (P = 0.018, Table 2).

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Figure 1 Expression of microRNA-218. A: Expression of microRNA (miR)-218 in the serum of gastric cancer patients (n = 68) and healthy controls (n = 56) (^aP = 0.026); B: Relationship between expression of miR-218 and tumor stages I/II (n = 40) and III/IV (n = 28) (^aP = 0.023); C: Relationship between expression of miR-218 and well-differentiated (n = 26) and poorly differentiated (n = 18) tumor (^aP = 0.025); D: Relationship between expression of miR-218 and tumor metastasis (n = 28) and non-metastasis (n = 40) (^aP = 0.031).

The relationships between miR-218 expression and clinicopathological variables of gastric cancer are summarized in Table 1. There was no significant relationship between miR-218 expression and patient age (P = 0.816), sex (P = 0.866), and tumor location (P = 0.916). However, miR-218 was found to be significantly associated with tumor metastasis (P = 0.003), tumor T stage (P = 0.018), and tumor grade (P = 0.012). Taken together, these findings strongly imply that miR-218 expression was negatively correlated with tumor differentiation, invasion, and metastasis, which are associated with tumorigenesis and tumor progression.

The expression level of miR-218 correlated with 3-year survival and tumor stage. Associations between the miR-218 expression parameters and overall survival were evaluated by Kaplan-Meier survival analysis with log-rank statistics for determining significance (Figure 2). The mean overall survival was significantly decreased in the low miR-218 group compared to the high miR-218 group (low miR-218: mean overall survival 23.6 mo, high miR-218: mean overall survival 35.6 mo; P = 0.008). For patients with high miR-218, 97% survived for 3 years after surgery, while for those with low miR-218 expression, only 54% survived. In patients with low, medium and high miR-218 expression, the percentage of those in stage I decreased by 87%, 31.5% and 17.5%, respectively. This implies that increased expression of miR-21 correlates with increased gastric cancer stage.

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Figure 2 Kaplan-Meier estimates of cumulative overall survival by microRNA-218. Significant difference was observed between the high and low microRNA (miR)-218 subgroups indicating the predictive value of miR-218 for gastric cancer (P = 0.008).

In a multivariate analysis based on the Cox proportional hazards regression model, we tested the independent predictive value for miR-218 expression as well as relevant clinical and pathological parameters including sex, age, tumor location, tumor size, metastasis or not, T stage and grade. As shown in Table 3, decreased miR-218 expression proved to be an independent prognostic marker for overall survival (P = 0.035), in addition to the presence of tumor metastasis (P = 0.005) and high tumor grade (P = 0.000).

Poor expression of miRNAs reportedly plays an important role in gastric carcinogenesis. microRNA-218 (miR-218) has been shown to be a tumor suppressor miRNA in several cancers. Furthermore, large microarray assays have indicated that there is significant downregulation of miR-218 in gastric cancer.

It was reported that miR-218 was downregulated in glioma, bladder cancer, lung cancer and oral cancer. In vitro assays showed that restored expression of miR-218 enhanced tumor growth and invasion, and reduced apoptosis. In the present study, the authors demonstrated expression of miR-218 in the serum of gastric cancer patients and its relationship with clinicopathological characteristics, tumor invasion and prognosis.

Large microarray assays have indicated that there is significant downregulation of miR-218 in gastric cancer. The status of miR-218 in the serum of gastric cancer patients has not been established. This is believed to be the first study about the expression of miR-218 in the serum of gastric cancer patients and its relationship with clinicopathological characteristics, tumor invasion and prognosis. Furthermore, this study suggests that miR-218 expression in serum is a prognostic marker in gastric cancer.

By understanding the expression of miR-218 in the serum of gastric cancer patients and its relationship with clinicopathological characteristics, tumor invasion and prognosis, this study may provide a future strategy for the treatment of patients with gastric cancer.

miRNAs were discovered as a novel class of evolutionarily conserved small (18-24 nucleotides) noncoding RNA molecules that are important regulators of gene expression. miRNAs are involved in cellular proliferation, differentiation, apoptosis, angiogenesis, invasion and migration. Several tumor suppressor miRNAs are reduced in human malignancies. miR-218 is one of these, and in vitro assays showed that restored expression of miR-218 enhances tumor growth and invasion, and reduces apoptosis.

This study was interesting, with a novel approach to the molecular biology of gastric cancer. The assay method was of good quality. The number of patients and controls was adequate. This was a carefully performed study and the findings are of considerable interest. This is perhaps the first report about the expression of miR-218 in serum of gastric cancer patients, which may be important for the diagnosis, treatment and prognosis of gastric cancer.