| Part I The expression and DNA methylation of Duoxl in hepatocellular carcinomaObjective:The gene Duoxl is a key phenotype of NADPH-oxidases. The main fuction of such genes is reactive oxygen species (ROS) production. Duoxl is highly expressed in thyroid, epithelial cells in airway while is lowly expressed in thyroid and lung cancer. In thyroid carcinoma, the expression of Duoxl is correlation with tumor malignant degree. So Duoxl is considered as a selective tumor suppressor gene. Furthermore, in lung cancer, Duoxl is predominantly silenced by promoter hypermethylation. However, the role of Duoxl in liver tumorigenesis remains unknown. So the aim of the present study was to investigate methylation status and expression level of Duoxl in HCC and try to elucidate its regulation mechanisms. Moreover, to analyze the relationship of the expression of Duoxl and the malignant biological characteristics of HCC.Methods:Duoxl mRNA expression in HCC cell lines and 20 paired HCC specimens was measured by quantitative real-time PCR (RT-PCR). RT-PCR was also used to detect Duoxl mRNA expression level in HCC cells before and after application of demethylation agent 5-Aza-dC. The basal promoter region of Duoxl was identified by methylation specific PCR and unmethylation specific PCR.Results:1. The mRNA expression of Duoxl in 6/8 liver cancer cell lines was significantly lower than that in the normal liver cell line.2. Duoxl mRNA expression was significantly downregulated in 17/20 primary hepatocellular carcinomas using real-time PCR when compared with adjacent non-tumor tissues (P=0.001).The mRNA expression of Duoxl is negatively correlation with the histilogical grade of HCC, while it was no correlation with the TNM stage of HCC.3. When treated by demethylating agent 5-Aza-dC, the expression of Duoxl significantly upregulated in 6/8 liver cancer cell lines.4. Duoxl promoter methylation was hypermethylated in 6 human liver cancer cells with downregulation of Duoxl.5. Duoxl promoter hypermethylation was detected in 90% primary liver tumor tissues and it was detected in 25% adjacent non-tumor tissues, the result was statistically significant (P<0.0001).Conclusion:Our data indicate that promoter DNA methylation is a major mechanism for silencing of Duoxl in HCC. The gene Duoxl may play a tumor suppressor in the carcinogenesis.Part IIThe roles of Duoxl and its potential tumor suppression mechanism in HCCObjective:Duoxl, as a member of NOXs, is major intracellular non-mitochondrial sources of ROS. Increasing evidences show that concentration of ROS can act as a second celluer messenger, regulating cell proliferation, differentiation and apoptosis to limit tumor occurrence and development. It has been reported that in lung cancer, Duoxl plays a role of tumor suppressor gene by inducing H2O2 production, promoting cell migration and wound repair. But its role in HCC is still unknown. So this part of the study is aimed to elucidate the role of Duoxl and its potential mechanism of action in HCC in vitro.Methods:The function of Duoxl on tumor suppression was determined through colony formation assay, cell growth assay and cell cycle analysis in Duoxl over expressed and knockdown HCC cell lines.Results:1. Ectopic expression of Duoxl reduced·cancer cell colony formation abilities and inhibited cancer cells growth (P<0.05);2. Downregulation of Duoxl promoted cancer cells growth (P<0.05);3. Ectopic expression of Duoxl led to a significant increase in G2/M phase in liver cancer cell lines (P<0.05)Conclusion:Our results indicate that Duoxl is inactivated in HCC and ectopic expression of Duoxl may inhibit the growth of HCC cells through blocking the cell cycle process. |
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