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Objective To quantifiably measure the methylation frequency of 18 CpG sites in the 3′region of L1 gene and long control region(LCR) gene of HPVl6 DNA,and study the relationship between HPVl6 DNA methylation and severity of cervical lesions. Methods A total of 10 cases Normal/low?grade squamous intraepithelial lesion(Normal/LSIL),10 cases of high?grade squamous intraepithelial lesion(HSIL),and 10 cases of cervical cancer(CC)were recruited for the study. The relationship between severity of cervical lesions and HPV16 DNA methylation was analyzed by bisultlte?pyrosequencing. Results The methylation rate was highest in Normal/LSIL at position 7 089 located in 3′?L1,followed by CC. The low?est was found in HSIL. The difference in methylation percentage among the three lesions was significant(P=0.006). In 7 134,the proportion meth?ylation was also different among three groups(P=0.01),difference in methylation percentage between Normal/LSIL and CC,as well as Normal/LSIL and HSIL was significant(P=0.038,0.017). Conclusion The methylation status of CpG sites 7 089 and 7 134 in the 3′region of L1gene is asso?ciated with the severity of cervical disease. The quantification of HPV DNA methylation can be used for cervical disease screening in clinical samples.
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Objective: To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-γ) gene promoter and its effect on IFN-γ expression in chronic hepatitis B. Method: The authors recruited 30 patients with HBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequencing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood, mononuclear cells (PBMCs). The expression of IFN-γ was analyzed by real-time RT-PCR and ELISA. HBV DNA in PBMCs was detected by nested PCR. Results: The methylation level at CpG site -55 in the IFN-γ gene promoter was significantly increased, resulting in subsequent down-regulation of the expression of this cytokine in CHB. The methylation level at CpG site -55 was significantly higher in HBeAg-positive patients than in HBeAg-negative ones (P < 0.01) and was also significantly higher in PBMCs from HBV DNA-positive patients than from HBV DNA-negative ones (P < 0.01); the methylation level at CpG site -55 was positively correlated with the amount of HBV DNA in serum (P < 0.01). Conclusion: IFN-γ gene expression appears to be regulated by methylation of the IFN-γ gene promoter in CHB; the methylation level at CpG site -55 is associated with HBV infection.
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Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-7) gene promoter and its effect on IFN-7 expression in chronic hepatitis B. Method The authors recruited 30 patients with UBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequeneing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expression of IFN-γ was analyzed by real-time RT-PCR and ELISA. HBV DNA in PBMCs was detected by nested PCR. Results The methylation level at CpG site -55 in the IFN-γ gene promoter was significantly increased, resulting in subsequent down-regulation of the expression of this cytoldne in CHB. The methylation level at CpG site -55 was significantly higher in HBeAg-positive patients than in HBeAg-negative ones (P<0.01) and was also significantly higher in PBMCs from HBV DNA-positive patients than from HBV DNA-negative ones (P<0.01) ; the methylation level at CpG site -55 was positively correlated with the amount of HBV DNA in serum (P<0.01). Oonclusion IFN-γ gene expression appears to be regulated by methylation of the IFN-γ gene promoter in CHB; the methylation level at CpG site -55 is associated with HBV infection.
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OBJECTIVE: X-linked inhibitor of apoptosis (XIAP) is the most potent member of IAP family that exerts antiapoptotic effects by interfering with activities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. We explore the candidacy of XAF1, Smac/DIABLO and HtrA2 as a tumor suppressor in cervical carcinogenesis and determine the mechanisms of altered XAF1 expression. METHODS: We investigated the expression and mutation status of the genes in 64 cervical cancer tissues, 5 cervical cancer cell lines and 10 normal cervical tissues. RESULTS: XAF1 transcript was not expressed or extremely low levels in 40% (2/5) of cancer cell line and in 31% (20/64) of primary carcinomas whereas Smac/DIABLO and HtrA2 are normally expressed in all cells. As somatic mutations of the gene was not detected, expression of XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza-2-deoxycytidine treatment. Bisulfite DNA analysis for CpG sites in the promoter region revealed a strong association between CpG sites hypermethylation and gene silencing. CONCLUSION: XAF1 undergoes epigenetic silencing in a considerable proportion of cervical carcinomas by aberrant promoter hypermethylation rather than genetic alterations, and closely associated with reduced gene expression. Although additional studies are required to determine the biological significance of XAF1 inactivation, it will be valuable to examine the expression status of XAF1 could be a clinically useful marker for cancer treatment.