Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:88 http://www.jeccr.com/content/29/1/88 Page 5 of 10 Table 1 Comparison of clinicopathological variables dependent on Snail and Slug mRNA ratios Slug mRNA (mean ± SE) Snail mRNA (mean ± SE) mean age (yr) <65(15) >65(37) Gender 62.2 ± 32.3 70.6 ± 17.5 Histologic grading G1 (29) G2 (16) G3 (7) Histology Well(13) Mod.(27) Poor(12) Depth of invasion T1(8) T2(32) T3(12) Surgical margin involvement Negative (n = 38) Positive (n = 14) Nodal metastasis Negative (n = 32) Positive (n = 20) Lymphatic invasion Negative (n = 10) Positive (n = 42) Venous invasion Negative (n = 15) Positive (n = 37) Perineural invasion Negative (n = 12) Positive (n = 40) Distant metastasis Negative (n = 44) Positive (n = 8) Survival (mo) <12 (n = 9) >12 (n = 43) 86.9 ± 25.5 78.3 ± 19.7 62.2 ± 32.3 70.6 ± 17.5 66.4 ± 13.6 58.0 ± 26.56 73.2 ± 33.8 69.2 ± 18.4 76.0 ± 15.8 85.6 ± 29.2 79.2 ± 12.4 68.4 ± 19.7 80.2 ± 30.5 66.4 ± 16.7 77.6 ± 31.5 59.8 ± 23.3 77.4 ± 22.8 68.3 ± 10.9 75.6 ± 16.4 79.6 ± 30.7 87.2 ± 24.6 60.4 ± 16.8 52.9 ± 14.4 64.8 ± 19.6 146.3 ± 33.2 126.8 ± 24.5 103.3 ± 36.7 P 0.249 0.8488 0.7252 0.6109 0.3260 0.197 0.1448 0.0865 0.1015 0.3758 0.0747 0.5596 (Fig 3B). siRNA-Slug transfectants showed a remarkably increased expression of E-cadherin. (Fig 3A). The obser- vations provided direct evidence that Slug inhibition increased E-cadherin expression in human cholangio- carcinoma cells. Cell invasion detection We tested whether Slug knockdown affected the inva- sion capabilities of QBC939 cells by using an in vitro invasion assay. Cells were seeded in the upper part of a Matrigel-coated invasion chamber in a reduced (5%) FCS concentration. After 24 h, cells that migrated in the lower chamber containing a higher (10%) FCS concen- tration were stained and counted. In Slug-silenced cell lines, invasion was significantly reduced (Fig. 4A; P < 0.05). Compared with untreated cells, or mock-siRNA cells, no further decrease in invasion was observed . We also tested the effects of Slug overexpression on the invasion capability of FRH 0201 cells. Compared with data obtained using the parental cell lines, Slug P 0.1969 0.2415 0.2523 0.135 0.1962 0.2277 0.0102 0.1404 0.3524 0.134 0.0001 0.0443 149.3 ± 57.4 171.2 ± 62.8 127.4 ± 35.6 124.3 ± 71.8 107.2 ± 60.2 114.7 ± 53.5 125.4 ± 41.4 95.7 ± 28.3 108.4 ± 46.5 100.7 ± 31.1 117.1 ± 28.0 98.4 ± 34.6 109 ± 36.3 102.6 ± 49.4 124.8 ± 60.0 86.8 ± 75.6 109.8 ± 35.2 180.3 ± 49.4 154. 5 ± 40.1 120 ± 121.7 134.5 ± 30.6 155.2 ± 26.2 166.3 ± 40.4 163.8 ± 13.6 143.3 ± 27.5 176.5 ± 87.2 163.4 ± 54.4