Luminescence was quantified with a luminometer (Lumat CB 9507, Belthold, Wildbad, Germany). Image analysis IHC- or ISH-stained slides were examined by light microscopy using an Olympus BX50 (Olympus, Tokyo, Japan). Positive IHC results appeared light brown, brown, or dark brown, whereas positive ISH results caused the cytoplasm to appear blue. Slides were interpreted semi-quantitatively by selleck compound assessing the intensity and extent of staining on the entirety of each tissue section present on the slides, as described previously (Acs et al, 2003). Statistical analysis The comparison of GAC with adjacent non-tumour tissues for each member of the miR-17�C92 cluster, STAT3, and pSTAT3 were performed using Welch’s t-test.

The correlation between the miR-18a ISH staining score and the PIAS3, Survivin, Bcl-xL, and c-Myc staining scores were estimated using Spearman’s correlation coefficient by rank test. P-value<0.05 was considered to be statistically significant (*P<0.05, **P<0.01, ***P<0.001). Results Upregulation of miR-18a in human GAC tissues in comparison with adjacent non-tumour tissues The miR-17�C92 cluster (C13orf25) produces a single polycistronic primary transcript that yields six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a (Figure 1A). To determine the expression patterns of these six miRNAs, we quantified their expression levels by performing ISH assays on gastric TMA specimens. Our TMAs contained 73 GAC specimens and 10 adjacent non-tumour tissue specimens (Table 1). MicroRNA expression was defined by ISH staining scores, as described previously (Acs et al, 2003).

In this study, we found that expression levels of all miRNAs were increased in GAC tissues in comparison with adjacent non-tumour tissues: miR-17 (P<0.001), miR-18a (P<0.001), miR-19a (P=0.219), miR-20a (P<0.001), miR-19b (P<0.001), and miR-92a (P<0.001). MicroRNA-19a expression was the only member of the miR-17�C92 cluster that was not significantly differentially expressed between GAC tissues and adjacent non-tumour tissues (Figure 1B). We suspect that low stability, rather than differences in maturation or processing, caused the low abundance of miR-19a. The expression level of miR-92a was the highest among the members of the miR-17�C92 cluster, but the expression ratio of miR-18a in GAC vs adjacent non-tumour tissue was the highest (>20-fold) (Figure 1B and C).

We also quantified the miR-18a levels in gastric FFPE samples by qRT�CPCR analysis. In qRT�CPCR analysis, the efficiency of primer annealing influences the amplification of the product, making it difficult to compare expression levels of amplified products that use different primers. To allow us to compare the six miRNAs produced from a single polycistronic transcript, we calculated the Anacetrapib copy number for each of these six miRNAs, and normalised the values against a standard curve generated using individual synthetic miRNAs.