Epigenetic Changes Of Dact2 In Hepatocellular Carcinoma And Functional Study

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancer in our country. Its pathogenesis remains unclear. Epigenetic silence of wnt/β-catenin pathway associated genes plays important roles during HCC carcinogensis. Recent studies have demonstrated that Dact1 and Dact3 (homo sapiens dapper, antagonist of P-catenin) were epigenetic regulator of wnt/β-catenin pathway in HCC and colorectal cancer respectively. However, Dact2 epigenetic changes in HCC remains unclear.Aims1. To detect Dact2 expression and DNA methylation in immortal liver cell line and HCC cell lines.2. To examine Dact2 expression in normal liver, HCC and adjacent normal tissues.3. To detect DNA methylation of Dact2 in HCC tissues.4. To investigate the relationship of Dact2 expression and DNA methylation.5. To study the effects of Dact2 on cell growth.6. To clarify the possible mechanisms of Dact2 on cell growth.Methods1. RT-PCR and Western blot were performed to detect Dact2 expression in immortal liver cell line and HCC cell lines before and after 5-aza-dc treatment.2. Methylation specific PCR (MSP) was used to detect DNA methylation of Dact2 in HCC cell lines and tissues.3. Immunohistochemisty was performed to examine Dact2 expression in normal liver, HCC and adjacent normal tissues.4. LipofectamineTM 2000 was used to transfect eukaryotic expression vector into HCC cell lines. FCM and G418 were used to select transient transfection cells.5. Colony formation assay and BrdU incorporation assay were used to detect cell growth of transfected cell.6. Apoptosis was analyzed by nuclear staining with Hoechst 33258.Results1. HepG2, SMMC7721, SNU449, SNU182, PLC-PRF-5 and LO.2 cell lines were supplied by Gastroenterology Department of PLA General Hospital. Dact2 expression was detected in Lo.2 and PLC-PRF-5, and down regulated in HepG2, SNU182 and SMMC7721. And Dact2 was silenced in SNU449. Promoter region methylation was revealed by MSP in HepG2, SNU449, SMMC7721 and SNU182 cells. Dact2 expression can be reversed in HepG2, SNU449, SMMC7721 and SNU182 cells after treatment with 5-aza-dc.2. Cytoplastic positive staing of Dact2 was found in normal, adjacent tissues and cancer tissues, but was downregulated in cancer tissues (P<0.001). Significant different of Dact2 expression was found among tumor differentiation levels (P<0.001). Dact2 methylation was exhibited in 23 (60.5%) of 38 HCC cases. Among 19 pairs of tissues,16 were found to lose Dact2 expression compared with their matched normal HCC tissues. Eleven of these 16 cases had Dact2 hypermethylation in tumors (68.75%).3. Reexpression of Dact2 resulted in significant suppression of long-term cell growth more than 2-folds by colonal formation assays. In BrdU incorporation assay, the percentage of positive cells nuclei is decreased to 36.49% compare with control group (51.96%) (P<0.05).4. Effects of Dact2 on HCC cell apoptosis. Dact2 has no influence on apoptosis in HepG2 and SNU449 cell lines.In conclusion, Dact2 expression is silenced by promoter region hypermethylation in HCC. Restoration of Dact2 expression was induced by 5-aza-dc treatment. Forced expression of Dact2 in methylated HepG2 cell line result in growth suppression, but no apoptosis changes was found in HepG2 cell.