Quantitative methylation analysis reveals distinct association between PAX6 methylation and clinical characteristics with different viral infections in hepatocellular carcinoma - PubMed (original) (raw)

Quantitative methylation analysis reveals distinct association between PAX6 methylation and clinical characteristics with different viral infections in hepatocellular carcinoma

Yu-Lueng Shih et al. Clin Epigenetics. 2016.

Abstract

Background: Related to genetic alteration, frequent promoter hypermethylation is also a contributing factor in the development of human cancers. Recently, we discovered numerous novel genes that were aberrantly methylated in hepatocellular carcinoma (HCC) by using Infinium HumanMethylation27 BeadChip array. We utilized a quantitative methylation-specific PCR (Q-MSP) system for the evaluation of PAX6 methylation in 29 normal controls and 160 paired HCC tissues and their adjacent non-tumor tissues. We verified the correlation between the methylation status of PAX6 and clinical characteristics with different viral status.

Results: Paired-box 6 promoter methylation was observed in 39.4 %, 15.6 %, and 3.4 % in primary HCCs, adjacent non-tumors, and normal control tissues, respectively. Methylation of the PAX6 promoter region in HCCs significantly increased compared with control tissues. PAX6 was frequently methylated in HCV-positive HCC tissues (61.3 %) and rarely methylated in HBV-positive (22.1 %) and double-negative HCC tissues (33.3 %).

Conclusions: Our data suggests that promoter hypermethylation of PAX6 is a common event in HCCs and the association of PAX6 methylation in clinicopathological features is divergent with different viral status.

Keywords: DNA methylation; HCV; Hepatocellular carcinoma; PAX6; Q-MSP.

PubMed Disclaimer

Figures

Fig. 1

Genes and the association of genes with diseases in HCC with different etiologic factors. The Venn diagram drawing the unique and overlapping genes in HBV-positive (HBV), HCV-positive (HCV), and double-negative (NBC) HCC (a). DAVID ontology (disease) for genes abnormally methylated in HBV, HCV, and NBC-HCC (b)

Fig. 2

Expression analysis and methylation analysis of PAX6 in cell lines. PAX6 and GAPDH expression were analyzed by the RT-PCR in the normal control, the normal liver cell line THLE-3, and the HCC cell lines treated with or without DNMT inhibitors (5-aza-2′-deoxycytidine; 5DAC) (a). Promoter methylation of PAX6 and COL2A was analyzed by MS-PCR with methylation-specific primers in the normal control, THLE-3, and HCC cell lines treated with or without 5DAC. CpG methylated human genomic DNA and DNA extracted from normal peripheral blood lymphocytes (PBL) which were modified by sodium bisulfite to generate a positive control, 1/5 diluted positive control, 1/20 diluted positive control, and a negative control, respectively (b). PAX6 methylation was also analyzed by bisulfite sequencing in Mahlavu cells treated with or without 5DAC. Each clone is represented by a row, and 30 CpG sites are represented as circles. Black and white circles represent methylated cytosine and unmethylated cytosine, respectively. Gray regions indicate the CpG sites that the MS-PCR/Q-MSP primer set covered (c)

Fig. 3

Methylation levels and ROC curve analysis of PAX6 in liver tissues. Gene methylation was determined in 29 normal controls (NC) and 160 paired hepatocellular carcinoma (HCC) tissues and their adjacent non-tumor tissues (NT) by Q-MSP. The results are represented as the difference in the methylation index. The black lines indicate the mean of the methylation index. The p value for the methylation levels among the groups was computed from Wilcoxon rank-sum test (NC vs HCC and NC vs NT) and Wilcoxon signed-rank test (NT vs HCC) (a). The best cutoff value for the methylation index (MI) and the area under the receiver operating characteristic curve (AUC) were calculated to discriminate 29 normal controls and 160 HCC (b). Gene methylation was respectively determined in normal controls (NC) and paired hepatocellular carcinoma (HCC) tissues and their adjacent non-tumor tissues (NT) by etiologic factors. The p value for methylation levels among the groups was computed from Wilcoxon rank-sum test (NC vs HCC and NC vs NT) and Wilcoxon signed-rank test (NT vs HCC) (c)

References

1. Bosch FX, Ribes J, Cleries R, Diaz M. Epidemiology of hepatocellular carcinoma. Clin Liver Dis. 2005;9(2):191–211. doi: 10.1016/j.cld.2004.12.009. - DOI - PubMed
1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010;127(12):2893–917. doi: 10.1002/ijc.25516. - DOI - PubMed
1. Arrieta O, Cacho B, Morales-Espinosa D, Ruelas-Villavicencio A, Flores-Estrada D, Hernandez-Pedro N. The progressive elevation of alpha fetoprotein for the diagnosis of hepatocellular carcinoma in patients with liver cirrhosis. BMC Cancer. 2007;7:28. doi: 10.1186/1471-2407-7-28. - DOI - PMC - PubMed
1. Nguyen VT, Law MG, Dore GJ. Hepatitis B-related hepatocellular carcinoma: epidemiological characteristics and disease burden. J Viral Hepat. 2009;16(7):453–63. doi: 10.1111/j.1365-2893.2009.01117.x. - DOI - PubMed
1. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128(4):683–92. doi: 10.1016/j.cell.2007.01.029. - DOI - PMC - PubMed

Quantitative methylation analysis reveals distinct association between PAX6 methylation and clinical characteristics with different viral infections in hepatocellular carcinoma - PubMed (original) (raw)