Written informed consent was obtained from each patient before tissue acquisition. All data were collected in the Department of Anatomical Pathology, Afflited hospital of Qingdao medical college, Qingdao university (Qingdao, China) from July 2000 to Sep. 2008. All tumors were defined as EHC, and pathological features of the tumors were determined histologically based on classifications of the Liver Cancer Study Group of China . Histological grades of the tumors consisting of more than two features were defined by the most prominent feature, and those components were selected for immunohistochemical studies. Real-Time Quantitative RT-PCR of Snail and Slug Total RNA was extracted

and purified from 52 paired samples of fresh frozen cancerous tissues and noncancerous bile tissues using Trizol Reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. For reverse transcriptase reaction, we used 5 μg of the RNA, random BIBW2992 hexamers, and Superscript II reverse transcriptase (Life Technologies, Inc.) according to the manufacturer’s instructions. The oligonucleotide primers and

TaqMan probes designed for Snail and Slug were as follows: Snail (5′-ACCACTATGCCGCGCTCTT-3′ and CFTRinh-172 supplier 5′-GGTCGTAGGGCTGCTGGAA-3′); Slug (5′-TGTTGCAGTGAGGGCAAGAA-3′ and 5′-GACCCTGGTTGCTTCAAGGA3′); and TaqMan probe (Snail, 5′-6FAM-TCGTCAGGAAGCCCTCCGACCC-TAMRA-3′ and Slug, 5′-6FAM-AGGCTTCTCCCCCGTGTGAGTTCTAATG-TAMRA-3′). Each primer was placed in a different exon to avoid amplification of contaminating Idasanutlin genomic DNA. Primers and probe for GAPDH (TaqMan GAPDH control reagent kit) were purchased

from Perkin-Elmer Applied Biosystems (Foster City, CA). Real-time quantitative PCR was done using the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems), as described above. Real-time PCR assays were done in triplicate, and the mean values were used for calculations of mRNA expression. Finally, the Snail and Slug mRNA expression ratios for tumorous (T) and nontumorous (N) tissues were calculated as follows: R = [Snail or Slug (T)/GAPDH (T)]/[Snail or Slug (N)/GAPDH (N)] × 102. Cases Cepharanthine were designated as either overexpression (R > 100) or nonoverexpression (R ≤ 100) cases. Immunohistochemical Staining of E-Cadherin Formalin-fixed, paraffin-embedded tissue sections from 52 EHC cases that corresponded to the RNA extracted cases were processed for immunohistochemical staining, as described previously [23] . A primary monoclonal Ab against E-cadherin (diluted 1:1000; Transduction Laboratories) was used. Positive immunoreactivity of normal bile duct epithelium was confirmed as a positive control for each specimen [24] . Immunohistochemical staining was examined under a light microscope by two pathologists. The cell staining of E-cadherin was evaluated semiquantitatively, and tumors were divided into two groups: (a) preserved pattern: >75% of tumor cells staining and (b) reduced pattern: <75% of tumor cells staining, as described elsewhere [23] .