| Background and objectiveHepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide. Each year, at least 300,000 people die from HCC in China alone. In China, risk factors for HCC include chronic hepatitis B or C infection, liver cirrhosis, and nonalcoholic steatohepatitis, et al. As for many other cancers, HCC has been understood a set of disease that driven by progressive genetic abnormalities and epigenetic changes of suppressor genes. The CpG islands hypermethylation of tumor suppress genens has been recognized as an important and alternative mechanism in hepatocarcinogenesis. Aberrant activation of the canonical Wnt/p-catenin signaling pathway is frequently involved in a broad spectrum of human cancers including HCC. AXIN2 gene is a tumor suppressor, as a scaffold protein, it plays an important role in the canonical Wnt/β-catenin signaling pathway. Epigenetic silencing of AXIN2 by promoter methylation underlies an alternative mechanism in lung and colon cancers development. However, it is currently unknown whether AXIN2 hypermethylation is associated with the HCC carcinogenesis. We investigate the promoter methylation status and mRNA expression of AXIN2 gene in HCC, and analyze the correlation of HCC and AXIN2 methylation status. Materials and Methods53 HCC and adjacent non-cancerous tissues (> 2cm away from tumor) of cirrhotic liver,17 normal liver tissues were surgically obtained from the patients in Henan province tumor hospital between May 2009 and April 2010. Most of the patients (48/53) had chronic hepatitis B infection. The age of the 53 patients (48 male, 5 female) ranged from 35 to 76 years, with a median age of 52 years.16 patients are BCLC stage A,4 patients are BCLC stage B,33 patients are BCLC stage C, no patient is BCLC stage D. Informed consent was given by all patients for investigations and data anaylsis. All patients were not treated by preoperative radiotherapy or chemotherapy. As a control,7 samples of normal liver tissues were collected from the resection of hemangioma. All cases were confirmed by pathologic diagnosis.Human HCC cell lines (HepG2,SMMC-7721,Skhep-1,Hep3B,PLC/PRF/5) were provided by Department of Microbiology, Peking University Health Science Center.RNA was extracted from HCC tissues and cell lines. The purity of RNA was determined by OD260/OD280. cDNA synthesis was performed using Fermentas Reverse Transcription System. The expression of AXIN2 mRNA was determined by real-time quantitative RT-PCR. DNA was digested by proteinase K and later was extracted through a phenol/chloroform treatment. Real-time PCR was used to calculate the methylation status of AXIN2 gene. Statistical tests were done using SPSS17.0 statistical software. The level for a statistically significant difference was set at a=0.05 for all the tests.Results1. The expression of AXIN2 mRNA was lower in HCC tissues than that in adjacent non-cancerous tissues (Z=-2.567,P＝0.010)2. In comparison with normal liver tissues, the methylation level of AXIN2 gene in HCC and adjacent non-cancerous tissues both increased significantly (t=-3.663, P=0.009; t=-4.591,P=0.007). 3. AXIN2 gene was also heavily methylated in all HCC cell lines. In contrast, the AXIN2 transcript was missing in all of the five HCC cell lines.4. AXIN2 mRNA levels were inversely correlated with DNA methylation (r=-0.458, P=0.032).5. Statistical analysis further indicated that AXIN2 hypermethylation was positively associated with BCLC stage.ConclusionsIn our experiment, decreased expression and hypermethylation of AXIN2 gene was found in HCC. AXIN2 mRNA levels were inversely correlated with DNA methylation. Methylation may be the main mechanism of AXIN2 gene down-regulation in HCC tissues, epigenetic silencing of AXIN2 may involve in the carcinogenesis of HCC.Hypermethylation of AXIN2 gene may affect the invasion and metastasis of HCC. |
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