MicroRNA-203 inhibits cell proliferation by repressing ΔNp63 expression in human esophageal squamous cell carcinoma

Human ESCC cell lines Eca109 and TE-1 were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were maintained in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin, within a humidified atmosphere containing 5% CO₂ at 37°C.

1 × 10⁶ cells cultured in a well of 6-well cell culture plate were transiently transfected with 50 pmol of miR-203 double-stranded mimics (or control microRNA) and ΔNp63 siRNA oligonucleotide duplexes (or control siRNA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol, respectively. Transfection efficiency was optimized using 6-carboxyfluorescein-labeled microRNA (or siRNA) at approximately 80% in Eca109 and TE-1 cells.

The sequences of miR-203 were:

Sense: 5'-GUGAAAUGUUUAGGACCACUAG-3',

Anti-sense: 5'-CUAGUGGUCCUAAACAUUUCAC-3',

A scrambled microRNA with no homology to any known human microRNA was used as negative control:

Sense: 5'-GUUGAACUGUUAAGAACCACUGG-3',

Anti-sense: 5'-CCAGUGGUUCUUAACAGUUCAAC-3',

The siRNA oligonucleotides targeting ΔNp63 were designed as previously described [25]:

Sense: 5'-AACAGCCAUGCCCAGUAUGUA-3';

Anti-sense: 5'- UACAUACUGGGCAUGGCUGUU-3'.

A scrambled siRNA with no homology to any known human mRNA was used as negative control:

Sense: 5'-CCCUGUUAAAAAUCCAGGCGA-3';

Anti-sense: 5'-UCGCCUGGAUUUUUAACAGGG-3'.

All microRNA mimics or siRNA oligonucleotide duplexes were synthesized by Genephama Biotech (Shanghai, China).

Total RNA was extracted from 1 × 10⁵ cells using the RNeasy RNA Mini Kit (Qiagen). First strand cDNA was synthesized using powerscipt reverse transcriptase (Clontech). The following gene-specific primer pairs were used for quantitative PCR:

Reverse, 5'-GAGTGGAATGACTTCAACTTT-3'.

Reverse, 5'-AGTTGGTGGTGCAGGATGC-3'.

PCR was performed using a Fast Start Master SYBR Green Kit (Roche) on a LightCycler (Roche). The expression level of ΔNp63 mRNA was analyzed using RealQuant software (Roche) and normalized to that of GAPDH mRNA.

Cellular proteins were prepared using cell lysis buffer (50 mM Tris-HCl, pH 8.0, 1% NP-40, 2 mM EDTA, 10 mM NaCl, 2 mg/ml aprotinin, 5 mg/ml leupeptin, 2 mg/ml pepstatin, 1 mM DTT, 0.1% SDS and 1 mM phenylmethylsulfonyl fluoride). Equal amounts of protein (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) overnight at 4°C before subsequent incubation with horseradish 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 β-actin protein.

Single-cell suspension was prepared using trypsin treatment. 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.

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)/(logN_t - logN₀), where t = time in culture (hr), N_t = final cell count, N₀ = original cell count.

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 distribution 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.

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.

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 cotransfected 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 β-Gal vector using Lipofectamine 2000. The Luciferase activity was examined at 48 hr posttransfection using the luciferase assay kit (Clontech) and normalized to β-galactosidase activity.

Data are presented as mean ± SEM. Statistical significance was tested using SPSS11.0 software, using t tests for 2-group comparisons. A P value less than 0.05 is considered statistically significant.

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 transfected with control microRNA (Figure 1A-C and Additional file 1, Table. S1). Meanwhile, cells transfected with miR-203 exhibited significantly lower colony forming efficiency as well as significantly longer population doubling time than those transfected with control microRNA (Figure 1D-F). These results suggest that miR-203 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 of cells (Eca109 and TE-1) transfected with ΔNp63 siRNA (or control siRNA). At 48 hr posttransfection, the expression of ΔNp63 protein and mRNA in the cells transfected with ΔNp63 siRNA was significantly decreased in comparison with that of cells transfected with control siRNA (Figure 2A-C), indicating that the expression of ΔNp63 was effectively inhibited by ΔNp63 siRNA. Subsequent studies showed that the proliferative capacity of cells transfected with ΔNp63 siRNA was significantly lower than that of cells treated with control siRNA. As shown in Figure 2d and 2e, cells transfected with ΔNp63 siRNA showed a significantly lower proliferative index as well as a significantly higher apoptotic index than those cells treated with control siRNA. Meanwhile, cells transfected with ΔNp63 siRNA exhibited a significantly lower colony forming efficiency as well as a significantly longer population doubling time than the control cells (Figure 2F and 2G). These results imply that repressing ΔNp63 expression could inhibit the proliferation of ESCC cell.

It was reported that the 3'-UTR of ΔNp63 contains the miR-203 binding site [14]. To determine whether the 3'-UTR of ΔNp63 mRNA is a functional target of miR-203 in ESCC cells, we evaluated the reporter activity in cells cotransfected with miR-203 (or control microRNA) and Luc-ΔNp63 plasmid (or Luc-ΔNp63-mut plasmid). As shown in Figure 3A, cells cotransfected with miR-203 and Luc-ΔNp63 plasmid showed a significant decrease of reporter activity in comparison with those cotransfected with the control microRNA and Luc-ΔNp63 plasmid. However, cells cotransfected with miR-203 and Luc-ΔNp63-mut plasmid showed no significant difference in reporter activity as compared with cells cotransfected with control microRNA and Luc-ΔNp63-mut plasmid (Figure 3B).

We further detected the expression of ΔNp63 protein and mRNA by western blot and qRT-PCR in Eca109 and TE-1 cells transfected with miR-203 (or control microRNA). As shown in Figure 3C and 3D, the expression of ΔNp63 protein was decreased by approximately 80% in cells transfected with miR-203 as compared to the cells treated with control microRNA at 48 hr posttransfection (0.08 ± 0.02 vs. 0.62 ± 0.10 for Eca109, P < 0.05; 0.13 ± 0.03 vs. 0.80 ± 0.11 for TE-1, P < 0.05). However, the expression of ΔNp63 mRNA showed no significant difference between the 2 groups (Figure 3E). These results indicate that the 3'-UTR of ΔNp63 mRNA is a functional target of miR-203 in ESCC cells.

To investigate the functional connection between miR-203 and ΔNp63 in the regulation of ESCC cell proliferation, we further evaluated the proliferative capacity of cells cotransfected with miR-203 and pcDNA-ΔNp63 (or empty pcDNA) plasmid. Notably, the pcDNA-ΔNp63 plasmid was designed to carry the open reading frame of human ΔNp63 without 3'-UTR. At 48 hr posttransfection, western blot revealed that the expression level of ΔNp63 in cells cotransfected with miR-203 and pcDNA-ΔNp63 plasmid was significantly higher than that in cells cotransfected with miR-203 and empty pcDNA plasmid (Figure 4A and 4B). Subsequent studies showed that the cells cotransfected with miR-203 and pcDNA-ΔNp63 plasmid had a significantly higher proliferative index, as well as a significantly lower apoptotic index than those cotransfected with miR-203 and empty pcDNA plasmid (Figure 4C and 4D). Meanwhile, cells cotransfected with miR-203 and pcDNA-ΔNp63 plasmid exhibited a significantly higher colony forming efficiency, as well as a significantly shorter population doubling time than the control cells (Figure 4E and 4F). These results imply that re-expressing ΔNp63 could significantly attenuate the inhibitory effect of miR-203 on cell proliferation, suggesting that the miR-203 inhibits the proliferation of ESCC cells through the ΔNp63-mediated signal pathway.