All reactions were performed in triplicate using the ABI 7300 real-time PCR system (Life Technologies). The ITGB6, B4 and A3 expression levels were normalized using the average expression levels of the endogenous control genes B2M and TBP. The correlations among the clinicopathological findings and among 3-MA molecular weight integrins β6, β4 and α3, fibronectin and laminin expression in CoCC, CCC, HCC and classical CHC were assessed by Fisher’s exact test or the

χ2-test and Mann–Whitney U-test. anova was used in the comparison among the ITGB6, B4 and A3 mRNA levels in the hepatic tumors. P < 0.05 was considered significant. IMMUNOHISTOCHEMISTRY DEMONSTRATED THE absence of β6 integrin in normal liver cells and bile duct epithelia but frequent expression on the bile duct epithelium of interlobular and septal ducts with weak positivity on bile ductular epithelium in injured liver tissues, including chronic hepatitis. No positivity for β6 integrin immunostaining was observed in 21 (91%) of 23 CoCC (Table 2, Fig. 1a,b,h) and all HCC (Fig. 1g),

whereas low or highly positive staining for these integrins was demonstrated in 23 (82%) of 28 CCC (Table 2, Fig. 1d–f). The cytoplasmic, cell membrane and basal lamina U0126 patterns of positive immunostaining were found in CCC (Fig. 1i) and CoCC. The predominant pattern observed in CCC was the cell membrane pattern (61%), whereas the cytoplasmic pattern or basal lamina pattern was predominant in each of two CoCC with focal positivity (Fig. 1c). Positive immunostaining for biliary type integrins learn more β4 and α3 in normal liver was evident on the biliary epithelium of the interlobular and septal

bile ducts, with faint or no positive staining on the bile ductular epithelium. Positive immunostaining for β4 and α3 on the bile duct epithelium was enhanced in the injured or diseased liver tissues. With regard to hepatic tumors, no or low positive staining for β4 was observed in most (91%) CoCC and all HCC, except one, whereas highly positive staining was evident in most (96%) CCC (Table 2, Fig. 2a–d,j). The predominant pattern of positive staining in CCC was the basal lamina type (Fig. 2d,k). Highly positive immunostaining for α3 was found in 11 (48%) of 23 CoCC and eight (19%) of 42 HCC, but it was more frequently observed in 21 (75%) of 28 CCC (Table 2, Fig. 2f–i,l). The immunoreactivity for α3 was localized to the cytoplasm of the tumor cells, and it was not detected in the cell membrane or basal lamina (Fig. 2f,h,i).