Yuan et al. BMC Cancer 2011, 11:57 http://www.biomedcentral.com/1471-2407/11/57 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 sig- nificantly 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 prolif- erative index as well as a significantly higher apoptotic index than those cells treated with control siRNA. Meanwhile, cells transfected with ΔNp63 siRNA exhib- ited 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. MiR-203 posttranscriptionally down-regulates ΔNp63 expression by targeting the 3’ untranslated region of ΔNp63 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 cotrans- fected with the control microRNA and Luc-ΔNp63 plas- mid. However, cells cotransfected with miR-203 and Luc-ΔNp63-mut plasmid showed no significant differ- ence in reporter activity as compared with cells cotrans- fected with control microRNA and Luc-ΔNp63-mut plasmid (Figure 3B). We further detected the expression of ΔNp63 pro- tein 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. Page 5 of 10 The inhibition of the proliferation of ESCC cells by miR- 203 could be significantly attenuated by the re- expressing of ΔNp63 To investigate the functional connection between miR- 203 and ΔNp63 in the regulation of ESCC cell prolifera- tion, 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 posttransfec- tion, 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 signifi- cantly higher colony forming efficiency, as well as a sig- nificantly 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, suggest- ing that the miR-203 inhibits the proliferation of ESCC cells through the ΔNp63-mediated signal pathway. Discussion It was known that microRNAs could regulate a variety of cellular pathways by affecting the expression of multiple types of target genes and the alteration of microRNAs expression might contribute to human carcinogenesis [9,11,12]. Thus, an understanding of the specific micro- RNAs involved in the process of tumor development would provide valuable insight for the diagnosis and treatment of patients with tumor. Here, we have demonstrated that miR-203 could down-regulate the proliferation of ESCC cells, probably through the ΔNp63-mediated signal pathway. Our data suggest that re-expressing miR-203 might benefit the treatment of ESCC. It was reported that the expression of miR-203 was sig- nificantly down-regulated in some tumors, including head and neck squamous cell carcinomas [14], hemato- poietic malignancy [13] and colon cancer [15]. Moreover, it was reported that inhibition of miR-203 expression could significantly increase the proliferation of Hela cells [26], whilst re-expression of miR-203 could inhibit the proliferative capacity of cells in human head and neck squamous cell carcinoma [14], hepatocellular carcinoma [16], chronic myelogenous leukemia and B cell leukemia