Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:88 http://www.jeccr.com/content/29/1/88 Page 3 of 10 cycles consisting of 15 s at 95°C, and a 1 min at 60°C. Serial 1:10 dilutions of plasmid DNA were analyzed for each target cDNA, and these served as standard curves from which we determined the rate of change of threshold cycle values. The amount of target gene expression was calculated from the standard curve, and quantitative normalization of Slug cDNA in each sam- ple was done using GAPDH as an internal control. Subcloning of Human Slug cDNA and Construction of Expression Plasmids The full coding region of human Slug was amplified by PCR using primers (5′-GCTGTAGGAACCGCCGT GTC-3′ and 5′-ATTTGTCATTTGGCTTCGGAGTG-3′) from cDNA of human EHC, and the product was cloned into the pT7 Blue vector (Novagen, Madison, WI). Iso- lated DNA sequences were determined using a cycle sequencing procedure. Slug cDNA was then subcloned into the bicistronic expression vector pGEM-T -EGFP (Clontech, Palo Alto, CA), which allows for translation of both the genes of interest and the EGFP. Cell Culture and Transient Transfection of Slug cDNA FRH 0201 cells were cultured at 37°C in 5% CO2 in RPMI 1640 (Life Technologies, Inc., Rockville, MD), containing 10% FBS (Life Technologies, Inc.). FRH 0201 cells (1 × 106) were grown in 3.5-cm dishes and transi- ently transfected with 2 μg of the pSlug-EGFP plasmid, as well as the empty pEGFP (mock) plasmid using Lipo- fectamine (Life Technologies, Inc.), according to the manufacturer’s instructions. At 48 h after transient transfection, Slug siRNA-transfected cells, which expressed both Slug and EGFP, were confirmed by epi- luminescence fluorescence microscopy (Axioscop2, Zeiss, Germany) . Small interfering RNA (siRNA) for inhibition of slug expression Three stealth small interfering RNA (siRNA) duplex oligoribonucleotides specific for Slug were synthesized. The sequences were as follows: 1) sense 5′-UUAACAGCAAACUCAGUUGAAAU GG-3′, antisense 5′-CCAUUUCAACUGAGUUUGCUGUU AA-3′; 2) sense 5′-UGAAUUAGGAAACUGAUCUUCC GGA-3′, antisense 5′-UCCAGAAGAUC AGUUUCCU AAU UCA-3′; 3) sense 5′-AAAUCUUUCAUGAUGAUUCCCU CGG-3′, antisense 5′- CCGAGGGAAUCAUGAAAGAUU U-3′. siRNA oligos were transfected into cholangiocarcinoma cells by using BLOCK-iT transfection kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The BLOCK-iT fluorescent oligo that is not homologous to any known genes was used as transfection efficiency detector and a negative control to ensure against induc- tion of non-specific cellular events caused by introduc- tion of the oligo into cells. Among the three siRNA oligo duplexes specific for slug, the one that required the smal- lest concentration to achieve the desired knockdown effect was selected and used in all experiments. Real-time RT-PCR for E-cadherin mRNA after transient transfection of Slug siRNA siRNA oligos were transfected into QBC939 (the highest level of Slug expression) cells (2 × 105) by using BLOCK-iT transfection kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol for 48 h. The mRNA inhibiting levels were assayed with Real-time RT-PCR . Tumor invasion in Matrigel-coated chambers To determine invasive ability, siRNA-Slug , Slug cDNA or mock control cells (1.25 × 105 per well)were plated on the BD Matrigel invasion chambers (BD Biosciences). Medium in the upper chamber was supplemented with 5% FCS. In the lower chamber, FCS concentration was 10%. After 24 h, cells migrated into the lower chamber were stained and counted. Experiments were carried out in triplicate and repeated twice. Statistical Analysis Follow-up was obtained through office records, tele- phone contact, or E-mail. Patient follow-up was com- plete up to September, 2008. Survival was calculated from the date of resection to one year after postopera- tion. All results were expressed as mean ± SE. Compari- sons between Snail/Slug expression levels (R; > 100 or ≤ 100) and E-cadherin expression patterns were evaluated using c2 test, and comparisons between the Snail/Slug expression ratios and clinicopathological parameters were evaluated using t test or F test. P of < 0.05 was considered to have statistical significance. Results Expression of Slug and Snail mRNA in extrahepatic hilar cholangiocarcinoma We quantified the copy numbers of Slug and Snail mRNA in 52 pairs of EHC tissue and noncancerous bile