The sections then were sequentially incubated with primary mouse antibodies against E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution, 1:150; monoclonal antibody) overnight at 4��C, a biotinylated goat anti-mouse secondary antibody (Santa Cruz Biotechnology) for 30 min, and peroxidase-conjugated streptavidin for 10 min. Samples kinase inhibitor Baricitinib were colored with diaminobenzidine (Boster, Wuhan, China) and counterstained with hematoxylin. Images were captured under an Olympus-BX50F4 microscope (Olympus Corporation, Tokyo, Japan) and quantitatively analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, USA). Sections of normal colorectal tissue were used as positive controls for E-cadherin staining. Negative controls were prepared by replacing the primary antibody with nonimmune IgG.

Statistical analysis The distributions of the clinical characteristics of the CRC patients and normal controls were analyzed by an unpaired two-tailed t-test. This test was also used to compare E-cadherin expression between the G-allele and GA-allele. The ��2-test was used to test the differences in the allele frequencies between normal controls and CRC patients. The genotype data were further stratified by age, sex, smoking, alcohol intake, tumor location, pathologic grouping and clinical stage of CRC. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model to evaluate the risk of the CDH1 -347G��GA polymorphism for CRC. We performed all analyses with the SPSS 15.0 software package (SPSS Inc, Chicago, USA).

Values of P < 0.05 were considered statistically significant. RESULTS All the recruited subjects were successfully genotyped. The study included 290 CRC patients and 335 normal controls with available data. The clinical characteristics of the study subjects are summarized in Table Table1.1. No significant differences were noted in the distribution frequencies for sex, smoking and drinking. As shown in Table Table2,2, the G-allele and GA-allele frequencies were 52.8% and 47.2%, respectively, in the CRC patients, and 57.9% and 42.1%, respectively, in the controls. There was no significant difference between the patients and the controls (��2 = 1.671, P = 0.198). A logistic regression analysis revealed that the GA-allele did not increase the risk of CRC (OR = 1.232, 95% CI = 0.898-1.691). However, when the CRC patients were stratified by age, sex, smoking, drinking, tumor location, pathologic grouping and clinical stage, the GA-allele frequency was higher in poorly differentiated CRC patients than in normal controls (��2-test, P = 0.002). In addition, the GA-allele frequency was higher in proximal CRC patients than in normal Carfilzomib controls (��2-test, P = 0.019).