Circulating microRNAs in plasma of patients with gastric cancers

Abstract

Background: We examined plasma microRNA (miRNA) concentrations from patients with gastric cancers (GCs) to assess their clinical application for diagnosing and monitoring diseases.

Methods: We initially investigated the appropriateness of plasma miRNA assay, and then compared plasma miRNA results with the expressions in cancer tissues from eight GC patients, and also compared plasma miRNAs between pre- and post-operative paired samples from 10 GC patients. Then, plasma miRNAs (miR-17-5p, miR-21, miR-106a, miR-106b and let-7a) were analysed in 69 GC patients and 30 healthy volunteers in total.

Results: The initial analysis showed that miRNAs were stable and detectable in all plasma samples, and the plasma miRNA levels reflected the tumour miRNAs in most cases. The levels of these miRNAs were significantly reduced in post-operative samples. In large-scale analysis, the plasma concentrations of miRNAs (miR-17-5p, miR-21, miR-106a, miR-106b) were significantly higher in GC patients than controls (P=0.05, 0.006, 0.008 and <0.001 respectively), whereas let-7a was lower in GC patients (P=0.002). The values of the area under the receiver-operating characteristic curve were 0.721 for the miR-106b assay and 0.879 for the miR-106a/let-7a ratio assay.

Conclusion: Detection of circulating miRNAs might provide new complementary tumour markers for GC.

Figure 1

Standard curve of miR-21, miR-106b, let-7a using synthetic miRNAs. Ten-fold serial dilution of synthetic miRNA was used to generate the standard curves. Linearity was confirmed within these concentrations, ranging from 1 to 0.0001 fmol. (miR-21: y=−3.7544x+14.318 (R²=0.999); miR-106b: y=−3.9849x+15.645 (R²=0.998); let-7a: y=−3.4988x+14.875 (R²=0.999))

Figure 2

Plasma miRNAs concentration in the initial analysis. Real-time RT-PCR assay, circulating plasma miRNAs (A: miR-21, B: miR-106b and C: let-7a) were detectable and amplified in all samples from 34 gastric cancer patients and 15 healthy volunteers. The concentrations of miR-106b and let-7a were significantly higher and lower in plasma from gastric cancer patients than in that from healthy controls (P=0.002 and <0.001 respectively). However, there was no significant difference in the concentration of miR-21 between gastric cancers patients and controls although it tended to be higher in gastric cancer patients (P=0.088). The upper and lower limits of the boxes and the lines inside the boxes indicate the 75th and 25th percentiles and the median respectively. The upper and lower horizontal bars denote the 90th and 10th percentiles respectively.

Figure 3

Comparison of plasma miR-21 and miR-106b concentrations between pre- and post-operative samples from gastric cancer patients. Expressions of both miRNAs were significantly reduced in plasma samples obtained 1 month after surgical removal of the tumour.

Figure 4

Box plots of the plasma miRNA concentrations in gastric cancer patients and controls. Plasma miRNA concentrations were significantly higher for miR-17-5p (P=0.05), miR-21 (P=0.006), miR-106a (P=0.008) and miR-106b (P<0.001) in the gastric cancer patients compared to those in controls, whereas let-7a was significantly lower in gastric cancer patients (P=0.002) . The upper and lower limits of the boxes and the lines inside the boxes indicate the 75th and 25th percentiles and the median respectively. The upper and lower horizontal bars denote the 90th and 10th percentiles respectively.

Figure 5

Receiver-operating characteristic (ROC) curve analysis in the concentration of miR-106b assay for detecting gastric cancer patients.

Figure 6

Receiver-operating characteristic (ROC) curve analysis of the ratio of miR-106a/let-7a assay for detecting gastric cancer patients.