To our knowledge, there have been no studies investigating a poss

To our knowledge, there have been no studies investigating a possible correlation among selleck bio the expression of DACT2, the clinicopathological factors in HCC, and the relevance of DACT2 expression to oncogenesis. In this study, we explored DACT2 gene expression in HCC and its possible association with clinicopathological factors. In addition, the epigenetic regulation of DACT2 in HCC cells was also analyzed. Methods Study population and tissue samples Sixty one HCC patients who had undergone liver trans plantation during 2003 and 2005 at our hospital were enrolled in this study. The study was approved by the local ethics committee, and informed consent was obtained from all of the patients. Of these patients, HCC was diagnosed either before or after transplantation, which was confirmed by histo pathological examination.

Complete clinical and labora tory data were Inhibitors,Modulators,Libraries available before operation and during follow up, and all patients were HBV positive. Distribution of clinicopathological data in the study cohort Inhibitors,Modulators,Libraries is given in Table 1. An additional 30 patients with HCC were also included Inhibitors,Modulators,Libraries in the present study. Specimens of cancer tissues and adjacent noncancerous tissues were collected from these patients after obtaining informed consent. Reverse transcription PCR and real time reverse transcription PCR Total RNA was extracted from hepatic specimens or cell lines, and cDNA was synthesized from total RNA. The messenger RNA expression level of theDACT2 was determined by RT PCR. The polymerase chain reac tion primer sequences for the DACT2 mRNA amplification have been described previously.

Real time PCR reactions were performed by the ABI7500 sys tem and SYBR green dye. Immunohistochemistry Immunohistochemical studies of Inhibitors,Modulators,Libraries DACT2 were performed on surgical specimens from HCC patients with cancerous and noncancerous adjacent tissues. Primary rabbit poly clonal antibodies against DACT2 were used at a dilution of 1 600. Immunohistochemistry was performed as described previously. Cell lines Liver cancer cell lines SMMC 7721, HepG2 and Hep3B as well as the metastasis capable human HCC cell lines MHCC97L and HCCLM3 were purchased. All cell lines were maintained in the recommended culture con dition and incubated at 37 C in a humidified environ ment containing 5% CO2. Bisulfite modification of DNA Genomic DNA was extracted from cell lines using the QIAamp DNA Mini Kit.

Inhibitors,Modulators,Libraries DNA samples were modified using EZ DNA Methyla tion Golden Kit. Bisulfite converts unmethylated CpG sites to UpG without modi fying methylated sites. Demethylation new with the DNA demethylating agent 5 Aza 20 deoxycytidine Liver cancer cells were plated at a density of 5 104 cellsml. After 24 h, cells were treated with 10 umoll of the DNA demethylating agent 5 aza 20deoxycytidine for 96 h. DNA and RNA were then extracted from cells.

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