AIM: To examine the methylation levels of interleukin-1 receptor-associated kinase 3(IRAK3) and GLOXD1 and their potential clinical applications in hepatocellular carcinoma(HCC).METHODS: m RNA expression and promoter methylation of IRAK3 and GLOXD1 in HCC cells were analyzed by reverse transcription-polymerase chain reaction(RTPCR) and methylation-specific PCR(MSP), respectively. Using pyrosequencing results, we further established a quantitative MSP(Q-MSP) system for the evaluation of IRAK3 and GLOXD1 methylation in 29 normal controls and 160 paired HCC tissues and their adjacent nontumor tissues. We also calculated Kaplan-Meier survival curves to determine the applications of gene methylation in the prognosis of HCC.RESULTS: IRAK3 and GLOXD1 expression was partially restored in several HCC cell lines after treatment with 5-aza-2′-deoxycytidine(DNA methyltransferase inhibitor; 5DAC). A partial decrease in the methylated band was also observed in the HCC cell lines after 5DAC treatment. Using GLOXD1 as an example, we found a significant correlation between the data obtained from the methylation array and from pyrosequencing. The methylation frequency of IRAK3 and GLOXD1 in HCC tissues was 46.9% and 63.8%, respectively. Methylation of IRAK3 was statistically associated with tumor stage. Moreover, HCC patients with IRAK3 methylation had a trend toward poor 3-year disease-free survival(P < 0.05). CONCLUSION: IRAK3 and GLOXD1 were frequently methylated in HCC tissues compared to normal controls and nontumor tissues. IRAK3 methylation was associated with tumor stage and poor prognosis of patients. These data suggest that IRAK3 methylation is a novel prognostic marker in HCC. AIM: To examine the methylation levels of interleukin-1 receptor-associated kinase 3 (IRAK3) and GLOXD1 and their potential clinical applications in hepatocellular carcinoma (HCC). METHODS: m RNA expression and promoter methylation of IRAK3 and GLOXD1 in HCC cells were analyzed by reverse transcription-polymerase chain reaction (RTPCR) and methylation-specific PCR (MSP), respectively. Using pyrosequencing results, we further established a quantitative MSP (Q-MSP) system for the evaluation of IRAK3 and GLOXD1 methylation in 29 normal controls and 160 paired HCC tissues and their adjacent nontumor tissues. We also calculated Kaplan-Meier survival curves to determine the applications of gene methylation in the prognosis of HCC. RESULTS: IRAK3 and GLOXD1 expression were partially restored in several HCC cell lines after treatment with 5- aza-2’-deoxycytidine (DNA methyltransferase inhibitor; 5DAC). A partial decrease in the methylated band was also observed in the HCC cell lines after 5DAC treatme Using GLOXD1 as an example, we found a significant correlation between the data obtained from the methylation array and from pyrosequencing. The methylation frequency of IRAK3 and GLOXD1 in HCC tissues was 46.9% and 63.8%, respectively. Methylation of IRAK3 was statistically associated with tumor stage. Moreover, HCC patients with IRAK3 methylation had a trend toward poor 3-year disease-free survival (P <0.05). CONCLUSION: IRAK3 and GLOXD1 were frequently methylated in HCC tissues compared to normal controls and nontumor tissues. IRAK3 methylation was associated with tumor stage and poor prognosis of patients. These data suggest that IRAK3 methylation is a novel prognostic marker in HCC.