Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:88 http://www.jeccr.com/content/29/1/88 Page 4 of 10 duct tissues using a TaqMan probe on ABI Prism 7700 Sequence Detection System, as described above. The copy number of Slug, Snail and GAPDH mRNA ranged from 218.4 to 83096, 117.8 to 15262, and 1238.56 to 6287429, respectively. Slug and Snail expression were standardized using the expression of the GAPDH house- keeping gene as the internal control. The cancerous (T)/ noncancerous (N) ratio of mRNA (R) was then calcu- lated to determine Snail and Slug mRNA levels in each case. Slug mRNA levels in cancerous tissue ranged from 0.823 to 58.9 (mean ± SE: 13.8 ± 3.1) and that of non- cancerous tissue from 4.14 to 142 (mean ± SE: 39.6 ± 4.8). The ratio (R) of Slug ranged from 0.04 to 658 (mean ± SE: 63.4 ± 19.3). 18 (34.6%) of 52 examined samples were defined as cases overexpressing Slug mRNA. The Snail mRNA levels were from 2.18 to 342 (mean ± SE: 47.8 ± 13.02) in cancerous tissue and from 8.80 to 163 (mean ± SE: 62.45 ± 6.8) in noncancerous tissue. The ratios (R) of Slug ranged from 3.14 to 1049 (mean ± SE: 132 ± 38.6). 12 (23%) of 52 samples exam- ined were defined as cases overexpressing Snail mRNA. Relationship between Slug and Snail expression and clinicopathologic data The relationship between Slug and Snail expression and clinicopathologic features is summarized in Table 1. The mean Slug mRNA ratio was significantly higher in cases of nodal metastasis (59.8 versus 77.4, P = 0.0102)and distant metastasis (64.8 versus 146.3, P = 0.0001). Patients with increased Slug mRNA(9/52)survived signif- icantly shorter than those with reduced Slug mRNA expression (43/52) (P = 0.0443). Cases of lymphatic invasion and perineural invasion also had high Slug mRNA ratios compared with the cases without invasion, although there was no statistical significance because of the distribution of the ratio [76.5 versus 68.3 (P = 0.1404), 60.4 versus 54.9 (P = 0.134), respectively. There was no statistical significance of Snail expression on clinicopathological parameters. Among the 18 Slug overexpression cases, 13 cases (72.2%) showed portal vein invasion and 7 (38.9%) showed liver artery invasion, whereas there were only 7 (20.6%)with portal vein invasion and 2 (5.9%) with liver artery invasion in 34 cases of Slug nonoverexpression. In addition, 10/18 showed remarkably high Slug mRNA levels (R > 200), and these were all with portal vein invasion. E-cadherin protein expression in EHC samples with or without Snail/Slug mRNA overexpression Expression of E-cadherin protein was also analyzed immunohistochemically. E-cadherin was expressed in membrane and/or cytoplasm.19 of 52 EHCs (36.5%) had a reduced expression pattern (Fig. 1). These findings did not significantly correlate with clinicopathological fea- tures such as distant metastasis, portal vein invasion, and liver artery invasion. The relationship between Snail/Slug mRNA expression and E-cadherin protein expression patterns was then determined in the EHC samples. Slug mRNA overexpression significantly corre- lated with E-cadherin reduced expression (Table 2) . 13 (72.2%) of 18 cases overexpressing Slug showed a reduced E-cadherin expression pattern, whereas only 6 of 34 cases of Slug nonoverexpression (17.6%) had a reduced pattern, with a statistically significant difference (P = 0.0001). However, there was no significant correla- tion between Snail overexpression and E-cadherin expression (Table 2). Ectopic expression of Slug to down-regulate E-Cadherin expression in EHC cell lines E-Cadherin mRNA expression was examined in a panel of three cholangiocarcinoma cell lines QBC939, SK-Ch- 1, FRH 0201 by real-time PCR and results showed that the cell line FRH 0201 had the highest expression level of E-Cadherin mRNA and the lowest expression of Slug mRNA (Fig 2A). In this regard, the cell line FRH 0201 was chosen for the studies.. The cell line FRH 0201 was transiently transfected with either full length human Slug cDNA-GFP vector or the control empty GFP vector. 48 h after transfection, cells were lysed and processed for mRNA analysis. In Fig 2B, the green fluorescent color indicates FRH 0201 cells transfected with control empty GFP vector. Cells were counted on the photographs and the ratio between green fluorescent cells and total cell number was taken as transfection efficiency. The transfection efficiency was 43.6% 48 h after transfection. Slug transfectants showed a remarkably reduced expression of E-cadherin protein, whereas positive E- cadherin expression was observed in nontransfected FRH 0201 cells. On the other hand, E-cadherin expres- sion was homogeneously preserved in mock-transfected cells (Fig 2C). These observations provided direct evidence that Slug repressed E-cadherin expression in human cholangiocarcinoma cells. siRNA Slug increases E-cadherin expression Slug mRNA expression was examined in a panel of three cholangiocarcinoma cell lines QBC939, SK-Ch-1, FRH 0201 by real-time PCR and results showed that the cell line QBC939 had the highest expression level of Slug mRNA (Fig 3A). In this regard, the cell line QBC939 was chosen for the studies. The cell line QBC939 was transiently transfected with Slug siRNA oligos for 48 h by using BLOCK-iT transfection kit. Cells were lysed and processed for mRNA analysis. The transfection efficiency was 32.4% 48 h after transfection