Yuan et al. BMC Cancer 2011, 11:57 http://www.biomedcentral.com/1471-2407/11/57 high density of microRNAs (including about 12% of the known human microRNA gene), exhibited significantly down-regulated expression in some tumors such as head and neck squamous cell carcinomas [14], hematopoietic malignancy [13] and colon cancer [15]. Subsequent stu- dies showed that the expression level of miR-203 is inversely correlated with the capacity of cell prolifera- tion in human head and neck squamous cell carcinoma [14], hepatocellular carcinoma [16], chronic myelogen- ous leukemia and B cell leukemia [13]. In addition, ΔNp63, an important oncogene regulating cell prolifera- tion in some tumors [17], was recently identified as a target gene of miR-203 in human epithelial precursor cells, as well as human head and neck squamous cell carcinoma cells [14,18]. These findings suggested that there might be a functional connection between miR- 203 and ΔNp63 in cell proliferation regulation. In human ESCC, genome-wide microRNA expression profile assay showed that the expression level of miR- 203 was significantly down-regulated in tumor tissue compared with the matched normal tissue [19-21]. In contrast, the expression level of ΔNp63 was significantly higher in tumor tissue than in the matched normal tis- sue [22,23]. However, the effect of miR-203 and ΔNp63 on the proliferation of ESCC cells, as well as the func- tional relationship between miR-203 and ΔNp63 in ESCC cells has not been documented. Since certain microRNAs may show different functions in different type of tumors [24], the role of miR-203 in the regula- tion of cell proliferation of ESCC warrants investigation. In light of the previous reports, we hypothesized that miR-203 might regulate the proliferation of ESCC cells through the ΔNp63-mediated signal pathway. Therefore, using 2 human ESCC cell lines (Eca109 and TE-1) as a model system, here we set out to investigate the effect of miR-203 and ΔNp63 on the proliferation of ESCC cell, as well as the regulation of the expression of ΔNp63 by miR-203 in ESCC. Our results suggest that miR-203 may inhibit the cell proliferation in ESCC through the ΔNp63-mediated signal pathway. Methods Cell culture Human ESCC cell lines Eca109 and TE-1 were pur- chased 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. Cell transfection 1 × 106 cells cultured in a well of 6-well cell culture plate were transiently transfected with 50 pmol of Page 2 of 10 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). Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) 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: ΔNp63: Forward, 5’-GGGTGAGCGTGTTATT- GATGCT-3’; Reverse, 5’-GAGTGGAATGACTTCAACTTT-3’. GAPDH: Forward, 5’-GCTGAGTATGTCGTGGA GTC-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. Western blot 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