He et al. Journal of Experimental & Clinical Cancer Research 2011, 30:109 http://www.jeccr.com/content/30/1/109 Page 2 of 9 the CDK family so far, where these members have some homology to a certain extent. CDK has a catalytic subu- nit that is activated in the presence of a regulatory subu- nit provided by cyclin [7], which leads to the formation of a mediator complex together with MDE12 and MED13. The mediator complex can bind to RNA poly- merase II, which participates in eukaryotic gene tran- scription such as the transcription of the b-catenin signaling pathway. Taken together, CDK8 plays an important regulatory role in cell cycle control and cell growth at the transcription level and it is proposed to be a proto-oncogene in human colon cancer [8-10]. As far as we know, studies on the role of CDK8 in the pro- liferation, apoptosis and cell cycle progression of colon cancer cells are still insufficient [11]. RNA interference (RNAi) has emerged as a powerful tool to induce lose- of-function phenotypes by post-transcriptional silencing of gene expression [12,13]. In the present study, CDK8 specific interference was designed and transfected into a colon cancer cell line HCT116. The effect of small inter- fering RNA (siRNA) silencing of CDK8 on the growth of colon cancer cells was investigated. In addition, we veri- fied the mRNA and protein expression levels of CDK8 and b-catenin in colon cancer tissues. Methods Major reagents Rabbit anti-human CDK8 antibody, rabbit anti-human b-catenin antibody, and rat anti-human b-actin antibody were purchased from Chemicon (USA). Lipofectin2000 was provided by Invitrogen (USA). RT-PCR kits were purchased from Fermentas (USA). Annexin V apoptosis kit (Keygentec, China) and siRNA-CDK8 (Genepharma, China) were used in the present study. Cell culture The human colon cancer cell line, HCT116 cell line was purchased from Shanghai Cell Biology Institutes, Chi- nese Academy of Sciences (Shanghai, China). HCT116 cell line was seeded in 6-well plate at a density of 1.5 × 105/well and maintained in RPMI1640 (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS). All cells were cultured at 37°C in a humidified atmo- sphere containing 5% CO2. Transfection with CDK8-siRNA CDK8 siRNA sequence 5’-AUAUAAUAGUGACUU- CACCAUUCCCTT-3’ (S) 5’-GGGAAUGGUGAAGU- VAVUAUUAUAUTT-3’ (AS) and scrambled siRNA sequence 5’-UUCUCCGAACGUGUCACGUTT-3’ (S) 5’-ACGUGACACGUUCGGAGAATT-3’ (AS) were designed and synthesized by Genepharma (Shanghai, China). HCT116 cells (1.5 × 105) were divided into three groups: (a) siRNA-CDK8 group, (b) scrambled siRNA group, and (c) non-siRNA control group. One hour before transfection, the medium was replaced with 1.5 ml of serum free Opti-MEM. Then, the following reagents were mixed as follows: (a) 4 μl of Lipofec- tin2000 were added to 250 μl of Opti-MEM, and the mixture was kept at room temperature for 5 min; (b) 4 μl of siRNA stock solution were added to 250 μl of Opti-MEM (siRNA dilution); and (c) the above two mix- tures were then mixed and kept at room temperature for 20 min (siRNA-Lipofectin2000 mixture). After shak- ing, this siRNA-Lipofectin2000 mixture was then added to a 6-well plate (1.5 ml of Opti-MEM in each well). Six hours later, the medium was replaced with complete medium. Our previous study confirmed that we obtained the maximal transfection efficacy when the ratio of Lipofectin2000 to siRNA was 4 μl:4 μl. MTT assay Six hours after transfection, HCT116 cells were digested, re-suspended and seeded in a 96-well culture plate. After 24, 48 and 72 h of incubation, cells were stained with 20 μl 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenylte- trazolium bromide Methylthiazolyl tetrazolium (MTT) solution (5 mg/ml) at 37°C for 4 h and subsequently made soluble in 150 μl of DMSO. Absorbance (A) was measured at 490 nm with an automated plate reader. Each sample was triplicated and the experiment was repeated three times. Cell growth curves were calculated as mean values of each group. Flow cytometric analysis Cells were trypsinized and centrifuged at 1500 rpm/min for 5 min at 48 h after transfection. Cells were harvested and washed with Phosphate Buffered Saline (PBS) twice. Reagents for apoptosis detection were added, and then cells were incubated in dark for 30 min and subjected to flow cytometry analysis (FACS). Additionally, cells were collected, washed with PBS, fixed with 75% ethanol at- 20°C overnight, and centrifuged at 1500 rpm/min for 5 min. Then, ethanol was removed and cells were washed with PBS twice. Propidium iodide (PI) and 500 μl of RNAse were added, and then cells were incubated in dark at 4°C for 60 min. Lastly, cells were subjected to cell cycle analysis by FACS. Gene expression analysis (RT-PCR and real-time PCR) The mRNA expression of CDK8 and b-catenin in HCT116 cells after CDK8-siRNA transfection were quantified by RT-PCR. Total RNA was extracted from cells with Trizol and subjected to reverse transcription into cDNA. CDK8 and b-catenin were amplified from the cDNA by RT-PCR. The PCR conditions consisted of 5 min at 94°C one cycle, 30 s at 94°C, 40 s at 55°C, 45 s at 72°C, and 7 min at 72°C 40 cycles. The primer