Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:88 http://www.jeccr.com/content/29/1/88 Page 8 of 10 Figure 4 siRNA knockdown of Slug and overexpression of Slug with the invasive potential in EHC cells. Cells were seeded in the upper chamber in medium supplemented with 5% FCS. Results are reported as percent migration ± SD compared with untreated cells. Experiments were carried out twice in triplicate. A Slug silencing inhibits invasion potention of QBC939 cells in Matrigel-coated invasion chambers. B Slug overexpression promotes invasive potential in FRH 0201 cells in Matrigel-coated invasion chambers. We found significant E-cadherin reduction in Slug overexpression cases, however, there were 28 (82.4%) with reduced E-cadherin expression but without Slug overexpression. Kanai et al.[34] reported that 48% show DNA hypermethylation of the E-cadherin promoter region and 42% show loss of heterozygosity at the locus adjacent to the E-cadherin gene in HCC. Genetic muta- tion of the E-cadherin gene was detected in breast, gas- tric, and gynecological cancers, which showed a uniform loss of E-cadherin expression[35-37] . To date, a genetic mutation of the E-cadherin gene has not been reported in cases of EHC in which loss of E-cadherin expression is considered to be heterogeneous and reversible. There- fore, E-cadherin expression in EHC may be regulated not just by the Slug transcriptional factor but also by other genetic and/or epigenetic alterations such as DNA mutation and/or methylation. Additional studies are required to reveal the entire regulatory mechanism of E- cadherin expression in EHC tumors. In this study, Slug mRNA overexpression correlated with metabasis and invasion of surgically resected human EHC. High expression of Slug mRNA has signifi- cantly shorter survival, the expression of Slug mRNA in EHC is an independent poor prognostic factor. EHC is hence a useful marker for predicting the outcome of patients with EHC who had a surgical resection of the tumor. Our data show that Slug, rather than Snail, nega- tively regulates E-cadherin expression, but it may also regulate the expression of other genes involved in the invasive potential of EHC. E-Cadherin has been reported to involve in tumor invasiveness [38-42] , but the rela- tionships between E-cadherin and clinicopathological factors were not consistent among these studies. In this study, E-cadherin was not found to be related to any clinicopathological factors. Differences of etiology and methods of evaluation might cause this discrepancy [40-42] . Additionally, the reversibility of E-cadherin expression should be considered. Slug and other family proteins bind to specific target genes and function as transcriptional repressors, but it is considered that the repression of E-cadherin alone is not sufficient to explain the role of Slug in cell migration and cancer development. The possible involvement of rhoA, rhoB, and other molecules, as well as E-cadherin, in the Slug pathway that controls cell motility has been considered for Drosophila, Caenorhabditis elegans, and vertebrate [43-46]. Additional investigations are needed to fully understand the functions and target genes of Slug protein in EHCs. Acknowledgements We take this opportunity to specifically thank the reviewers and editors for their kind instructions that may be helpful for our further studies. Author details 1General Surgery, the Affiliated Hospital of medical College, Qingdao University, Qing dao, Shandong Province. 266003. China. 2Laboratorian, the Affiliated Hospital of medical College, Qingdao University, Qing dao, Shandong Province. 266003. China. 3Urinary surgery, the Affiliated Hospital of medical College, Qingdao University, Qing dao, Shandong Province. 266003. China. 4Anatomic Pathology, the Affiliated Hospital of Hainan Medical college, Haikou. 527010. China. Authors’ contributions ZKJ, WDS and ZSY designed the experiments. ZKJ and JXL carried out most of experiments and drafted the manuscript. WXS, YQC and CHN carried out the immunohischemistry and RT-PCR. LCW and WDS participated in statistical analysis and and interpretation of data. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Received: 25 February 2010 Accepted: 1 July 2010 Published: 1 July 2010