Methylation Of3-OST-2Gene In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma (HCC) is a common malignant tumor in China,the early diagnosis and molecular targeted therapy has become research hotspot. The carcinogenesis of HCC includes two mechanisms:genetic mechanism and epigenetic mechanism,which is a cascade control process,and eventually leading to the activation of oncogene or/and inactivation of suppressor gene. Epigenetics refers to gene expression changes which DNA sequence will not changed,the main contents of epigenetic mechanism is that DNA methylation and histone modification. In recent years, studies have shown that the occurrence and development of human tumor is related to abnormal DNA methylation, specific gene methylation detection can be used as molecular markers in early diagnosis of cancer, treatment targets and judgment of prognosis. The absence expression of heparan sulfate D-glucososaminyl3-O-sulfotransferase-2(3-OST-2) gene result in uncompletely modification of sulfuric acid acetyl glycoprotein, which has a close relationship with tumor development,but the specific mechanism is still unclear. DNA methylation is often interact with histone modification to regulate gene expression. Histone modifications of the regulation is more complex than DNA methylation, different histone amino acids can occur in a variety of different types of decoration, deacetylation and histone acetylation is the important content of histone modification.The recent research found that UHRF1not only can recognize methylation sites by DNA spiral enzyme and plays an important role in DNA replication, but also can identify methylated histone.UHRF1can double recognize DNA and histone methylation.But we remains unclear that how UHRF1regulate tumor-suppressor gene expression and play what role in HCC Objective:To investigate the function and mechanism of the promoter methylation status of heparan sulfate D-glucososaminyl3-O-sulfotransferase-2(3-OST-2) gene in hepatocellular carcinoma by detect the effects of methyltransferase inhibitor(5-Aza-dC) and hitone deacertylase inhibitor(TSA) on promoter methylation status of3-OST-2and nuclear protein UHRF1in human hepatocellular carcinoma.Methods:Develop HCC cell lines, the experiment was divided into four groups:(1) not dosing HCC cell line, control;(2) the individual treatment group,5-Aza-dC;(3) individual treatment group, the TSA;(4) combination treatment group,5-Aza-dC+TSA.The changes of promoter methylation of3-OST-2before and after the treatment of5-Aza-dC and/or TSA was detected by methylation specific PCR (MSP). The changes of mRNA and protein expression of3-OST-2or UHRF1were determined by real time RT-PCR, Western blotting and cell slide immunohistochemistry respectively.Results:3-OST-2complete methylation was detected in untreated BEL-7402cells and was partially reversed by5-Aza-dC or TSA, consistently, mRNA was increased by2.7times and4.9times respectively. After treatment by the combination of the inhibitors,3-OST-2methylation was completely reversed and mRNA was significantly increased by9.1times. UHRF1mRNA was decreased by5-Aza-dC or TSA(decreased77.45%and84.31%respectively). After treatment by the two drugs, mRNA was decreased90.20%.UHRF1protein was highly expressed in BEL-7402cells. After teated with5-Aza-dC or TSA, UHRF1protein was decreased (69%) and (35%).After treatment by the combination of the inhibitors, UHRF1protein was decreased (27%). Western blotting results indicated that5-Aza-dC or TSA can inhibits the expression of nuclear protein UHRF1, and the effect is more remarkable by the combination of the inhibitors.Conclusions:The decrease of3-OST-2gene plays an important role in HCC. Promoter methylation and histone modifications both caused the decrease of3-OST-2expression. UHRF1participated in interactions of DNA methylation and histone deacetylation of the3-OST-2gene expression regulation pathways, and has more regulatory effect of histone acetylation than DNA methylation.