Construction of IRX4-inducible system in pancreatic cancer cell lines, PK-1 and PK-9

Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to the loss of key cellular pathways in tumorigenesis. We have previously reported that IRX4 (Iroquois homeobox 4) was highly downregulated by promoter hypermethylation in pancreatic cancer cell lines as well as in resected primary pancreatic cancers. Based on these data, we have constructed a tetracycline-inducible IRX4 expressing system using pancreatic cancer cell lines PK-1 and PK-9. IRX4 induction significantly suppressed cell growth and caused apoptosis in both PK-1 and PK-9. Because IRX4 is a sequence-specific transcription factor, we tried to analyze IRX4 downstream events by performing microarray analyses using IRX4 inducible PK-1 cells with or without tetracycline. We found that IRX4 induction upregulated several genes that had tumor suppressive functions such as PRDM1, CRYAB, and IL32, and downregulated several genes that had oncogenic functions such as PTCH1, CD36, TFAP2C, and MUC4. These results suggest that DNA methylation-mediated silencing of IRX4 may contribute to pancreatic tumorigenesis through aberrant transcriptional regulation of cancer-related genes. Two pancreatic cancer-derived cell lines, PK-1_IRX4#28 and PK-9_IRX4#23, that allow tetracycline-regulated IRX4 expression were constructed. The RNAs from PK-1_IRX4#28 and PK-9_IRX4#23 with (4 days treatment) or without tetracycline were prepared and analyzed by using Agilent whole human genome microarray (4×44K) v2.