He et al. Journal of Experimental & Clinical Cancer Research 2011, 30:109 http://www.jeccr.com/content/30/1/109 Page 3 of 9 sequences were as follows: 5’-TCACCTTTGAAGCCTT TAGC-3’ (forward) and 5’-CTGATGTAGGAAGTGG GTCT-3’ (reverse) for CDK8; 5’-TGCCAAGTGGGTGG TATAGAG-3’ (forward) and 5’-TGGGATGGTGGGTG TAAGAG-3’ (reverse) for b-catenin; 5’CTGGGACGA CATGGAGAAAA3’ (forward) and 5’AAGGAAGGCTG GAAGAGTGC3’ (reverse) for b-actin. The mRNA expression of CDK8 and b-catenin in colon cancer samples (n = 12) were quantified by real- time PCR. Informed consent was obtained from all the patients, and research protocols were approved by Inde- pendent Ethics Committee (IEC) of our hospital. A total of 200 mg of tumor or adjacent normal tissues (at least 2 cm distant from the tumor site) were homogenized in liquid nitrogen. Total RNA was extracted and reverse transcribed into cDNA, which was then used for ampli- fication of CDK8 and b-catenin. The real time PCR con- ditions consisted of 1 cycle at 94°C for 10 min followed by 40 cycles at 94°C for 30 s, at 55°C for 30 s, and at 72°C for 30 s. GAPDH was employed as an internal standard. The primer sequences were as follows: 5’- GAGCGGGTCGAGGACCTGTTTGAAT-3’ (forward) and 5’-ACATGCCGACATAGAGATCCCAGTTCCT TC-3’ (reverse) for CDK8; 5’-TGCCAAGTGGGTGG TATAGAG-3’ (forward) and 5’-TGGGATGGTGGGTG TAAGAG-3’ (reverse) for b-catenin; 5’AGGGGCCATC- CACAGTCTTC3’ (forward) and 5’ AGAAGGCTG GGGCTCATTTG 3 (reverse) for GAPDH. The 2 -ΔΔCT method was applied to analyze the relative changes in gene expression. Western blot analysis As described previously [14], following 72 h of transfec- tion, total protein was extracted from HCT116 cells and subjected to SDS-PAGE. Protein concentrations were transferred onto PVDF membrane, then membranes were blocked and incubated with rabbit anti-human CDK8 (1:1000) or b-catenin antibody (1:1000) at 4°C overnight. After 3 washes with TBS-T solution for 10 min, the membranes underwent hybridization with a goat anti-rabbit IgG secondary antibody (1:1000) at 37°C for 1 h. After further washing, CDK8 and b-catenin levels were visualized using an ECL chemiluminescence kit. Immunohistochemistry The protein expression of CDK8 and b-catenin in 47 tumor tissues and adjacent normal tissues were detected by IHC. Samples were fixed in 10% neutral formalde- hyde, embedded in paraffin, and sliced. Briefly, the par- affin-embedded tissues were serially cut into 4 μm sections, dewaxed, and rehydrated. Sections were then blocked with peroxide and non-immune animal serum and incubated sequentially with rat anti-human CDK8 and b-catenin (1:1000), and biotin-labeled goat anti-rab- bit IgG (1:1000). Finally, the sections were stained with DBA, counterstained with hematoxylin, dehydrated, cleared in xylene, and fixed. Histological assessment was performed as described previously [15]. Immunostaining was independently examined by two clinical pathologists who were unaware of the patient outcome. Five high-power fields (400 × magnification) were randomly counted for each section. The brown staining on the cytoplasm was read as posi- tive reactivity for CDK8 and b-catenin. The presence of brown colored granules on the cytoplasm was taken as a positive signal, and was divided by color intensity into not colored, light yellow, brown, tan and is recorded as 0, 1, 2, 3, respectively. We also choose five high-power fields from each slice and score them. Positive cell rate of < 25% was a score of 1, positive cell rate of 25~50% was a score of 2, positive cell rate of 51~75% was a score of 3, positive cell rate of > 75% was a score of 4. The final score was determined by multiplying positive cell rate and score values: 0 was equal to negative ( - ), 1~4 was equal to weakly positive (+), 5~8 was equal to moderate positive(+ +), 9~12 was equal to strongly posi- tive (+ + +). Statistical analysis All data were presented as means ± standard deviation (SD). A Student’s t-test was used for comparisons between groups, and F test was applied for correlation analyses. Statistical analysis was performed with SPSS 13.0 statistic software package. P values < 0.05 were considered to be statistically significant. Results Effect of CDK8-siRNA transfection on CDK8 and b-catenin expression in HCT116 cells Six hours after CDK8-siRNA transfection, the transfec- tion efficiency was detected by FACS. Our previous study confirmed that the maximal transfection efficacy could be obtained when the ratio of Lipofectin 2000 to siRNA was 4 μL: 4 μL. (Figure 1) Forty-eight hours later of CDK8-siRNA transfection, RT-PCR was performed to detect CDK8 and b-catenin mRNA expression. The results showed that mRNA expression of CDK8 and b-catenin was markedly lower in the CDK-siRNA group compared with the other two groups (P < 0.01) (Figure 2). However, there was no sig- nificant difference in mRNA expression between the scrambled siRNA group and non-siRNA group. Following a 72 h CDK8-siRNA transfection of HCT116 cells, protein expression of CDK8 and b-cate- nin was determined by western blot assay. As shown in figure 3, CDK8 and b-catenin expression was remark- ably reduced in the CDK-siRNA group compared to the