Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:88 http://www.jeccr.com/content/29/1/88 Page 2 of 10 expression could be repressed by mechanisms other than promoter hypermethylation [8] . The heterogene- ity and reversibility of E-cadherin protein expression are both controversial areas [3]. Recently, the Slug transcription factor was reported to directly repress E-cadherin expression in many epithelial cancers asso- ciated with epithelial-mesenchymal transitions [12]. Reverse correlation of Slug and E-cadherin expression has been noted in many malignant cells[13-19]. It has reported that Snail, a zing-finger protein, is a likely repressor of E-cadherin in carcinoma Cells[20-22]. However, we can find no documentation regarding the expression of Snail or Slug in human EHC tissue. In this study, we investigated whether Slug represses E-cadherin expression in human EHC cells. The levels of expression a of Snail and Slug mRNA were detected in a series of human EHC samples, and correlations between Snail/Slug expression and clinicopathological factors were analyzed. Our evidence suggests that Slug, rather than Snail, may contribute to both E-cadherin expression and to the progression of EHCs. Materials and methods Patients This present retrospective study was based on data obtained using surgically resected tissues from 52 conse- cutive Chinese patients who underwent hepatectomy for EHCs. Written informed consent was obtained from each patient before tissue acquisition. All data were col- lected in the Department of Anatomical Pathology, Affl- ited 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 con- sisting 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 noncan- cerous bile tissues using Trizol Reagent (Life Technolo- gies, Inc.) according to the manufacturer’s instructions. For reverse transcriptase reaction, we used 5 μg of the RNA, random hexamers, and Superscript II reverse tran- scriptase (Life Technologies, Inc.) according to the man- ufacturer’s instructions. The oligonucleotide primers and TaqMan probes designed for Snail and Slug were as follows: Snail (5′-ACCACTATGCCGCGCTCTT-3′ and 5′-GGTCGTAGGGCTGCTGGAA-3′); Slug (5′-TGTT GCAGTGAGGGCAAGAA-3′ and 5′-GACCCTGGTTG CTTCAAGGA3′); and TaqMan probe (Snail, 5′-6FAM- TCGTCAGGAAGCCCTCCGACCC-TAMRA-3′ and Slug, 5′-6FAM-AGGCTTCTCCCCCGTGTGAGTTC- TAATG-TAMRA-3′). Each primer was placed in a dif- ferent exon to avoid amplification of contaminating genomic DNA. Primers and probe for GAPDH (Taq- Man 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 fol- lows: R = [Snail or Slug (T)/GAPDH (T)]/[Snail or Slug (N)/GAPDH (N)] × 102. Cases 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 evalu- ated 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] . Real-time RT-PCR for E-cadherin mRNA and Slug mRNA in EHC cell lines QBC939, SK-Ch-1, FRH 0201 cells, the cultured human EHC cell line, were supplied from Cell Resource Center, FUDAN University (Shanghai, China). These cells were cultured at 37°C in 5% CO2 in RPMI 1640, containing 10% FBS. Upon reaching 70% confluence cells were lysed into Trizol reagent (Gibco, UK) for mRNA extraction and evaluation of E-cadherin mRNA and Slug mRNA expression by Real-time quantitative RT-PCR. Real-time quantitative PCR was done using the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems) as described previously [23]. Briefly, each PCR mixture contained 1 μl of cDNA, TaqMan Universal PCR mas- ter mix (Perkin-Elmer Applied Biosystems), primer pair, and TaqMan probe in a final volume of 50 μl. The PCR conditions were an initial denaturation step of 2 min at 50°C and 10 min at 95°C, followed by 40