In addition to formalin fixation for routine histopathological diagnosis, fresh tumor tissues and,

when possible, noncancerous mucosal tissues distant from the TSCC lesion were collected immediately after resection, placed separately in an RNA stabilization regent (RNAlater, Qiagen, Valencia, CA), and stored at −80°C until further analysis. For this study, 40 patients were selected on the basis of the availability of frozen tissue from which RNA find more of sufficient quality could be extracted. The clinicopathological characteristics of the patients were collected from the medical records, and the tumor stages were classified according to the American Joint Committee on Cancer TNM staging system. We evaluated the histopathological characteristics of the tumor specimens (i.e.,

histological grade [differentiation], vascular invasion, lymphatic invasion, and perineural invasion) by reviewing each slide stained with hematoxylin and eosin. Statistical analysis The data obtained in the in vitro experiments are presented as mean ± standard deviation (SD). The mRNA LY411575 clinical trial expression levels of CDH1, SIP1, Snail, Twist, and Cox2 in the clinical samples are indicated as median values and ranges because of the skewed distribution of the data. Differences in the mRNA expression levels between paired samples (tumor vs. noncancerous) were assessed using the Wilcoxon signed Epacadostat rank-sum test. Correlations between the mRNA expression levels and clinicopathological factors were evaluated using the Mann-Whitney U-test or the Dipeptidyl peptidase Spearman rank

correlation coefficient. Risk factors of lymph node metastasis were examined using Fisher’s exact test, the chi-square test, or the Mann-Whitney U-test for the univariate analysis, and a multiple logistic regression model with the stepwise selection method for the multivariate analysis. P-values less than 0.05 were considered statistically significant. All statistical analyses were performed using SPSS Ver. 16.0. Results Baseline mRNA expression of Cox-2, CDH-1, and its transcriptional repressors in HNSCC Cells We used quantitative real-time PCR to evaluate the mRNA expression levels of Cox-2, E-cadherin transcripts (CDH-1) and its transcriptional repressors (SIP1, Snail, and Twist) in HNSCC cell lines. The relative expression levels of each gene were normalized by dividing each value by that of SAS cells as a calibrator for convenience. As shown in Figure 1A, a trend toward an inverse correlation was found between Cox-2 and CDH-1 by Spearman rank correlation coefficient (rs = −0.714, p = 0.055). HT-1080 cells showed no CDH-1 expression as expected as the negative control for E-cadherin. Figure 1B displays the relative expression levels of the transcriptional repressors. Interestingly, the expression level of SIP1 was revealed to be significantly correlated with that of Cox-2 (rs = 0.771, p = 0.042) and inversely correlated with that of CDH-1 (rs = −0.