The primers for GAPDH (224 bp) were 5′-TGAAGGTCGGAGTCAACGG-3′ (sense) and 5′- CTGGAAGATGGTGATGGGATT-3′ (I-BET151 datasheet antisense). The primers for L1CAM (187 bp) were 5′-TGTCCTTCCCTTTACGCCAC-3′ (sense) and 5′- GACCAAGCACAGGCATACAGG-3′ (antisense). The primers for EPCAM (101 bp) were 5′-ATAATAATCGTCAATGCCAGTG-3′ (sense) and 5′- ATTCATTTCTGCCTTCATCAC-3′ (antisense). The expression of GAPDH was used to normalize that of the target genes. Each assay was done in triplicate and the average calculated. The expression level of

L1CAM/EPCAM was expressed as 2–ΔΔCt, ΔCt = Ct(Target) – Ct(GAPDH). Tissue microarray Blocks containing a total of 693 cases (601 cancer samples and 92 non-cancer tissue samples) were prepared as described previously [1, 2]. Immunohistochemistry Immunohistochemical analysis selleck chemical was used to study altered

protein expression in 92 noncancerous human gastric tissue controls and 601 human gastric cancer tissues [3, 4]. In brief, slides were baked at 60°C for 2 h, followed by deparaffinization with xylene and rehydration. The sections were submerged into EDTA antigenic retrieval buffer and microwaved for antigenic retrieval, after which they were treated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity, followed by incubation with 1% bovine serum albumin to block nonspecific binding. Sections were incubated with rabbit anti-EPCAM(Epitomics), and with mouse anti-L1CAM (Abcam), overnight at 4°C. Normal goat serum was used as a negative control. After washing, tissue sections were treated with secondary antibody. Tissue sections were then counterstained with hematoxylin, dehydrated,

and mounted. Cytoplasm with L1CAM and MK0683 EPCAM was stained as buffy. The degree of immunostaining was reviewed and scored independently by two observers based on the proportion of positively stained tumor cells and intensity of staining [5–7]. Statistical analysis All statistical analyses were performed using SPSS16.0 software. Measurement data were analyzed using Student’s t test, while categorical Myosin data were studied using χ 2 or Fisher exact tests. Survival curves were estimated using the Kaplan–Meier method; the log-rank test was used to compute differences between curves. Multivariate analysis using the Cox proportional hazards regression model was performed to assess prognostic values of protein expression. Correlation coefficients between protein expression and clinicopathological findings were estimated using the Pearson correlation method. Statistical significance was set at P < 0.05. Results Expression of L1CAM and EPCAM mRNA in gastric tumor tissue and cell lines L1CAM and EPCAM mRNA were significantly upregulated in AGS, MKN-28, BGC-823, HCG-27, SGC-7901, 9811P and MKN-45 cell lines compared with the non-malignant gastric epithelial cell line GES-1 (P < 0.05, Figure 1, Figure 2). In 42 gastric tumor tissue samples and matched normal gastric mucosa, average expressions of L1CAM were 0.0403 ± 0.