7 miRNAs were up-regulated (the expression in the carcinoma group was more than twice as high as in the normal group). The differentially expressive miRNAs were listed in Table 3. Table 3 miRNAs differential expression in gastric cancer samples compared with the normal samples Down-regulation (19) P Value Up-regulation (7) P Value miR-9 0.0073 miR-518b XAV-939 in vitro 0.009 miR-433 0.0041 miR-26b 0.0147 miR-490 0.0142 miR-212 0.0329 miR-155 0.021 miR-320 0.0179 miR-188 0.019 miR-409-3b 0.0352 miR-630 0.024 miR-30a-5b 0.0164 miR-503 0.0102 miR-379 0.0158 miR-611 0.0151     miR-545 0.0241     miR-567 0.0173    

miR-575 0.0109     miR-197 0.024     miR-649 0.0157     miR-19b 0.017     miR-338 0.0184     miR-383 0.0267     miR-652 0.0183     miR-551a 0.0166     miR-370 0.0112     Detection of miR-433 and miR-9 expression by Quantitative Real-time PCR MiR-433 and miR-9 were remarkably down-regulated by microarray analysis in the carcinoma samples. qRT-PCR was used to detect the expressive level of miR-433 and miR-9 in 3 normal gastric tissues, 24 malignant tissues, SGC7901 and GES-1 cell lines. We found that miR-433 was down-regulated 83% in the

carcinoma PD-1/PD-L1 tumor LY2835219 clinical trial tissues compared with normal gastric tissues. MiR-433 was down-regulated 77.3% (P < 0.05) in SGC7901 compared with GES-1 cell lines (Figure 1A). MiR-9 was down-regulated 75% in carcinoma tissues compared with normal gastric tissues. MiR-9 was down-regulated 76.2% (P < 0.05) in SGC7901 compared with GES-1 cell lines

(Figure 1B). The results were consistent to the microarray analysis. Figure 1 MiR-433 and miR-9 expression in normal gastric tissues, 24 malignant tissues, SGC7901 and GES-1 cell lines. A, miR-433 was down-regulated 83% in the carcinoma tissues compared with normal gastric tissues and down-regulated 77.3% (P < 0.05) in SGC7901 compared with GES-1 cell lines. B, miR-9 was down-regulated 75% in carcinoma tissues compared with normal gastric tissues and down-regulated 76.2% (P < 0.05) in SGC7901 C-X-C chemokine receptor type 7 (CXCR-7) compared with GES-1 cell lines. Identification of miR-9 and miR-433 targets We were further interested in miRNA-regulated gene targets, which enabled us to understand miRNA functions. To explain the potential roles of miR-9 and miR-433 in carcinogenesis, we predicted the targets of miR-9 and miR-433 via the algorithms: TargetScan, PicTar, and miRanda. To confirm whether the predicted targets of miR-9 and miR-433 were responsible for their regulation, the presumed target sites were cloned and inserted at the downstream of the luciferase gene of pGL3. Direction of junction fragments was identified and plasmids including junction fragments of norientation were chose. In Figure (2A), we found a 430 bp fragment, and in Figure (3A), we found a 580 bp fragment. The results were consistent to the amplification of pGL3-control and junction fragments sequences, which demonstrated that the fragments were norientation. XbaI was used to digest the junction fragments, then, we did electrophoresis.