Yuan et al. BMC Cancer 2011, 11:57 http://www.biomedcentral.com/1471-2407/11/57 phenylmethylsulfonyl fluoride). Equal amounts of pro- tein (50 μg) were separated by 10% SDS PAGE and then transferred to nitrocellulose membranes (NY, USA) by electroblotting. The membranes were blocked with 5% BSA in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) for 1 hr, and then incubated with mouse anti-human ΔNp63 antibody (Santa Cruz) over- night at 4°C before subsequent incubation with horse- radish peroxidase-conjugated goat anti-mouse antibody (BD) for 1 hr at 37°C. Protein was visualized using enhanced chemiluminescence reagent (Santa Cruz). The expression level of ΔNp63 protein was analyzed using LabWork 4.0 program (UVP) and normalized to that of b-actin protein. Clonogenic assay Single-cell suspension was prepared using trypsin treat- ment. Cells were then seeded into 6-well cell culture plates (200 cells/well) and incubated for 2 weeks at 37°C. Then, cells were washed twice with PBS and stained with a mixture of 6.0% glutaraldehyde and 0.5% crystal violet for 1 hour at 37°C. The plates were air- dried at room temperature. Colony forming efficiency was calculated as the percentage of plated cells that formed colonies. Cell population doubling time Cells were plated into 6-well plates (1 × 104 cells/well) and cultured at 37°C. Cell population doubling time (PDT) was calculated using the following equation: PDT (hr) = (log2 × t)/(logNt - logN0), where t = time in cul- ture (hr), Nt = final cell count, N0 = original cell count. Cell cycle assay Cells were fixed in 70% ethanol for 2 hr at 4°C. After washing with PBS, cells were treated with RNaseA (50 μg/ml) and stained with propidium iodide (25 μg/ml) for 30 min at 37°C. Samples were analyzed using an FACSCalibur flow cytometer (BD Biosciences) and dis- tribution of cell-cycle phases was determined using Modfit Software (BD Biosciences). The proliferative index was calculated as the percentage of cells in S/G2/ M-phase. Apoptosis assay Cells were stained with annexin V-FITC and propidium iodide using the ANNEXIN V-FITC Kit (Beckman) according to the manufacturer’s protocol and subjected to flow cytometric analysis. Viable cells were unstained by annexin V or propidium iodide, early apoptotic cells were stained by annexin V but not propidium iodide, and late apoptotic cells were stained by annexin V and propidium iodide. The apoptotic index was calculated as the percentage of annexin V+/propidium iodide- cells. Page 3 of 10 Luciferase reporter assay The full-length 3’-UTR of ΔNp63 mRNA containing the miR-203 binding site was amplified by PCR (Forward: 5’-ggggagctcatataagaactcttgcagtct-3’; Reverse: 5’- gggaagcttggtgtacattcttctagaac-3’). Mutant ΔNp63 3’- UTR, which carried a substitution of four nucleotides (CTTT to GAAA) within the core binding sites of ΔNp63 3’-UTR (14), was obtained using overlapping extension PCR. Normal (or mutant) ΔNp63 3’-UTR was cloned into the SacI-HindIII site of the pMIR-REPORT luciferase vector (Biosystems) and named as Luc-ΔNp63 (or Luc-ΔNp63-mut). Then, 1 × 106 cells were cotrans- fected with 50 pmol of microRNA-203 (or control microRNA), 1 μg of Luc-ΔNp63 (or Luc-ΔNp63-mut) plasmid, and 1 μg of pMIR-REPORT b-Gal vector using Lipofectamine 2000. The Luciferase activity was exam- ined at 48 hr posttransfection using the luciferase assay kit (Clontech) and normalized to b-galactosidase activity. Statistical Analyses Data are presented as mean ± SEM. Statistical signifi- cance was tested using SPSS11.0 software, using t tests for 2-group comparisons. A P value less than 0.05 is considered statistically significant. Results MiR-203 could inhibit the proliferation of ESCC cell lines We scanned the basal expression levels of miR-203 in the ESCC cell lines by real time PCR. It was found that both Eca109 and TE-1 expressed very low level of miR-203 (Additional file 1, Figure S1). Therefore, we investigated the effect of miR-203 on cell proliferation using cells transfected with miR-203 (or control microRNA). As shown in Figure S1, mature miR-203 was highly expressed in cells transfected with miR-203 while the expression level of miR-203 was still very low in cells transfected with control microRNA 48 hr posttransfection. In addition, cells treated with miR- 203 had a significantly lower proliferative index and a significantly higher apoptotic index than cells trans- fected with control microRNA (Figure 1A-C and Additional file 1, Table. S1). Meanwhile, cells trans- fected with miR-203 exhibited significantly lower colony forming efficiency as well as significantly longer population doubling time than those trans- fected with control microRNA (Figure 1D-F). These results suggest that miR-203 could inhibit the prolif- eration of ESCC cell. Repressing ΔNp63 expression could inhibit the proliferation of ESCC cell To investigate the effect of ΔNp63 on the proliferation of ESCC cell, we evaluated the cell proliferative capacity