Sporadic CRC cases and controls were matched for age, sex, smoking and drinking history. Table 1 Clinical characteristics of the CRC patients and normal controls The study inhibitor was approved by the Institutional Review Boards of the Drum Tower Hospital. All the participants provided written informed consent at the time of recruitment and agreed to blood collection. DNA extraction Peripheral venous blood (5 mL) was drawn from each subject before they received surgery or chemotherapy, and was placed in tubes containing EDTA and stored at -70��C until analysis. Total genomic DNA was extracted using a purification kit (Promega, Madison, WI, USA) according to the manufacturer��s instructions. Genotyping analysis The CDH1 -347G��GA polymorphism was genotyped by the PCR-RFLP method.

A 447-bp fragment containing the -347G��GA polymorphism in the CDH1 promoter was amplified with the following primers: forward, 5′-GCCCCGACTTGTCTCTCTAC-3′; reverse, 5′-GGCCACAGCCAATCAGCA-3′. PCR amplification was carried out in a volume of 25 ��L containing 20 ng of genomic DNA, 1 ��L of primers, 20 ��mol/L each of the forward and reverse primers, 2.5 ��L of 10 �� PCR buffer (Mg2+-free), 2 ��L of dNTP, 1.5 ��L of MgCl2 and 0.5 U of Taq polymerase (TaKaRa Biotechnology, Dalian, China). The amplification was performed in a programmable thermal cycler (MWG Biotech AG, Ebersberg, Germany) as follows: 1 cycle of 95��C for 2 min; 35 cycles of 94��C for 1 min, 61��C for 1 min and 72��C for 1 min; and a final cycle of 72��C for 10 min. The PCR product was digested with BanII (TaKaRa Biotechnology) at 37��C overnight (Figure (Figure1A1A and andB).

B). After digestion, the products were separated by 3% agarose gel electrophoresis and stained with ethidium bromide (Figure (Figure1C).1C). GA/GA homozygous cases were represented by DNA bands of 332 and 116 bp. G/G homozygous cases were represented by DNA bands of 263, 116 and 68 bp. GA/G heterozygous cases displayed a combination of both alleles (332, 263, 116 and 68 bp). Figure 1 Genotyping analysis of the CDH1 -347G��GA polymorphism by PCR-RFLP analysis. A: Structural diagram of the restriction enzyme analysis for BanII; B: Schematic overview of the E-cadherin gene promoter PCR fragment and the locations of the BanII restriction … Immunohistochemistry CRC tissue samples were collected from CRC patients who underwent surgery.

Normal colon tissue samples were collected from outpatients via colonic biopsies during colonoscopy examination. Immunohistochemistry was carried out using the avidin-biotin-peroxidase complex method. The central region of CRC tissue samples and normal colon AV-951 tissue samples were cut into 4-��m sections, mounted on glass slides, deparaffinized and rehydrated by xylene and graded ethanol solutions. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 20 min at room temperature.