Yuan et al. BMC Cancer 2011, 11:57 http://www.biomedcentral.com/1471-2407/11/57 Page 7 of 10 Figure 3 MiR-203 posttranscriptionally regulates ΔNp63 expression by targeting the 3’-UTR of ΔNp63. (A, B) 1 × 106 cells (Eca109 or TE- 1) were cotransfected with 50 pmol of miR-203 (or control microRNA) and 1 μg of Luc-ΔNp63 (or Luc-ΔNp63-mut) plasmid, respectively. Luciferase reporter assay were performed at 48 hr posttransfection. Results showed that cells cotransfected with miR-203 and Luc-ΔNp63 plasmid exhibited a significant decrease of reporter activity in comparison with those cotransfected with the control microRNA and Luc-ΔNp63 plasmid (a). However, the reporter activity of cells cotransfected with miR-203 and Luc-ΔNp63-mut plasmid showed no significant difference with that of cells cotransfected with control microRNA and Luc-ΔNp63-mut plasmid (b). (C, D) Cells (1 × 106) of Eca109 and TE-1 were transfected with 50 pmol of miR-203 (or control microRNA), respectively. The expression level of ΔNp63 protein was detected by Western Blot at 48 hr posttransfection and normalized to that of b-actin. Results showed that the level of ΔNp63 protein was significantly decreased in cells transfected with miR-203 as compared to the cells transfected with control microRNA. Results showed that the expression level of ΔNp63 protein was significantly decreased in cells transfected with miR-203 as compared to the cells transfected with control microRNA. (E) Cells (1 × 106) of Eca109 and TE-1 were transfected with 50 pmol of miR-203 (or control microRNA), respectively. The expression level of ΔNp63 mRNA was detected by qRT-PCR at 48 hr posttransfection and normalized to that of GAPDH. Results showed that the expression level of ΔNp63 mRNA exhibited no significantly difference between cells transfected with miR-203 and those transfected with control microRNA. Data represent mean ± SEM from 4 independent experiments; **, P < 0.01 by t test. Previous studies indicated that ΔNp63, an alternative splice variant of p63 gene lacking TA domain [17], is the major isotype expressed in a variety of human squa- mous cell carcinoma including ESCC [27], and that the expression level of ΔNp63 in tumor tissues was signifi- cantly higher than in the matched normal tissues [22,27]. In our pilot study, we found ΔNp63 was highly expressed, whilst TAp63 was hardly detectable in Eca109 and TE-1 (Additional file 1, Figure S2). Here, we demonstrated that repressing ΔNp63 expression by siRNA could significantly inhibit the proliferation of ESCC cell lines, implying that ΔNp63 played a positive role in ESCC cell proliferation. Our findings, in combination with the previous reports that ΔNp63 could promote the cell proliferation in head and neck squamous cell carcinoma [14] as well as lung squamous cell carcinoma [27], suggest that ΔNp63 may function as an oncogene in human squamous cell carcinoma. Recently, using bioinformatic analysis, Lena et al. [14] and Yi et al. [18] independently reported that the 3’- UTR of ΔNp63 contain the miR-203 binding site. Subse- quent studies showed that miR-203 could repress the expression of ΔNp63 and inhibit cell proliferation in human epithelial precursor cells as well as human head and neck squamous cell carcinoma cells, suggesting that miR-203 is a key molecule controlling the ΔNp63-