Three spliced transcript variants, HOPX, B, and, encode the same protein, which contains a putative homeodomain motif that acts as an adapter protein to mediate transcription. HOPX expression Ivacaftor Sigma is ubiquitous in wide arrays of normal tissue, but not in malignant tissues including choriocar cinoma, lung, uterine endometrial, and gastrointestinal cancers. The inactivation mechanism ac tually involves promoter methylation in esophageal, endometrial, and gastric cancer. Also, enforced HOPX expression inhibited tumor growth and RNA interference knockdown of endogenous HOPX restored it. These findings suggest that the HOPX gene acts as a tumor suppressor gene. In this study, we for the first time studied methylation level of HOPX gene in PC and added the functional assay to answer the question whether HOPX plays an important role in pancreatic carcinogenesis.

Methods Cell lines and tissue samples The pancreatic cancer cell lines, PK 8, KLM 1, and NOR P1 were kindly provided from the Cell Resource Centre for Biomedical Research Institute of Develop ment, Aging and Cancer, Tohoku University. Six other cell lines, PK 59, PK 45 H, PK 45P, MIA Paca2, PANC 1, or the esophageal squamous cell carcinoma cell line TE15 and gastric cancer cell line KatoIII were purchased from RIKEN BioRe source Centre. All cell lines except MIA Paca2 were maintained in RPMI 1640 Medium and MIA Paca2 was maintained in DMEM, containing 10% fetal bovine serum. Clinical tissue samples were categorized according to TNM classification, 7th edition of the Union Internation ale Contre Le Cancer and the 6th edition of the Japan Pancreas Society.

The patients characteris tics were depicted in Additional file 1 Table S1. All tis sue samples were collected at the Kitasato University Hospital, and informed consent was obtained. The present study was approved by the Ethics Committee of the Kitasato University. Bisulfite treatment of DNA and sequencing analysis Genomic DNA from homogenized bulky tissues and cell lines was extracted using QIAamp DNA Mini Kit. Bisulfite treatment was done by using an EpiTect bisulfite kit and the DNA was applied to polymerase chain reaction. PCR primer sequences were designed using DNA sequences converted by bisulfited treatment. The PCR products were sequenced using a Big DyeW Terminator v3. 1 Cycle Se quencing Kit.

For the clonsed sequence analysis, the PCR products were inserted into pCR4 TOPO vector using a TOPO TA cloning kit for sequencing, selected 15 clones for each sample and then sequenced. Quantitative methylation specific PCR TaqMan methylation specific PCR was carried out using iQ Supermix in triplicate on the iCycler iQTM Real Time PCR Detection system. PCR conditions and the primer sequences are pro vided in Table 1. Serial dilutions of bisulfite modified DNA from KatoIII Carfilzomib were used as positive control and TE15 as negative control, respectively.