So, it is an urgent need to reveal the underlying mechanisms by which pancreatic AZ 960 905586-69-8 cancer cells become invasive and metastatic. Hedgehog signaling cascade is aberrantly activated in a variety of human tumors including pancreatic cancer. The activation of Hh pathway requires the binding of Hh ligands, such as Shh, Ihh and Dhh, to Hh receptor Patched, thus releasing Hh signaling molecule Smoothened from Ptchinduced inhibition. Smo in turn initiates the release of the transcription factor GLI, thus facilitates its nuclear translocation, GLI activators then bind to the GACCACCCA-like motif for the transcriptional regulation of Hedgehog target genes, which are involved in the regulation of cellular proliferation, cell-fate determination, cellular survival, and epithelial-to mesenchymal transition and etc. A membrane glycoprotein Human Hedgehog Interacting Protein can bind to all three Hh ligands and functions to negatively regulate the activity of Hh signaling pathway. DNA methylation change is a key contributor to human oncogenesis. In human cancer cells, the normal somatic pattern of DNA methylation is altered. These changes include increased CpG island methylation, which mediates tumor suppressor gene silencing, and genomic DNA hypomethylation, which can lead to genomic instability. Cytosine DNA methylation is catalyzed and regulated by a small family of DNA methyltransferases, including DNMT1, DNMT3a, DNMT3b and DNMT3L. Although cancer-specific mutations of DNMTs have not been reported, several studies suggest that DNMT genes are overexpressed in human cancer and during cellular transformation. Several mechanisms seem to account for DNMTs overexpression, including aberrant cell cycle control, increased mRNA and protein stability, and E2F-mediated DNMTs promoter activation. Although the evidences above indicate that DNMTs and active Hh signaling pathway are both involved in the development of pancreatic cancer, little is known about the correlation between DNMTs and members of the Hh pathway. Here, this study was undertaken to investigate the expression of GLI1 and DNMTs, and the correlation between them in human pancreatic cancer. In this study, we found that GLI1, DNMT1 and DNMT3a are over-expressed in PC tissues compared with the corresponding noncancerous pancreas tissues, then we showed that DNMT1 and DNMT3a expression changed according to the GLI1 expression in PANC-1 and BxPC-3 cell lines by specific GLI1 interference and gene transfection, as well as pharmacological method in vivo. More importantly, we proved beyond a reasonable doubt that GLI1 was able to bind to the DNMT1 gene promoter of site 3 by the ChIP experiments. Finally, we used nested MSP to demonstrate that GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC. To the best of our knowledge, this is the first report demonstrated GLI1 as a transcriptional factor that regulated DNMT1 and expression as well as APC methylation level in PC, and DNMT1 is its direct target gene.