Methylation Profiling of Multiple Tumor Suppressor Genes in Hepatocellular Carcinoma and the Epigenetic Mechanism of 3OST2 Regulation

Abstract

DNA methylation is considered as a significant mechanism that silences tumor suppressor genes (TSGs) and could be used in the early diagnosis of cancer. Histone modifications often work together with DNA methylation; however, how these epigenetic alterations regulate TSGs remains unclear. Here, we determined the methylation status of ten TSGs (3OST2, ppENK, CHFR, LKB1, THBS1, HIC1, SLIT2, EDNRB, COX2, and CLDN7) in hepatocellular carcinoma (HCC) and corresponding noncancerous tissues. Methylation profiling revealed that four genes had very high frequencies of methylation in HCCs, but interestingly, similar high frequencies were also detected in corresponding noncancerous tissues (97.9% vs 95.8% for SLIT2, 93.8% vs 81.3% for EDNRB, 66.7% vs 85.4% for HIC1, and 56.3% vs 56.3% for ppENK, P > 0.05). Only the 3OST2 gene was frequently methylated in HCCs and there was significant difference between HCCs and corresponding noncancerous tissues (68.8% vs 37.5%, P < 0.05). 5-aza-2'-deoxycytidine (5-Aza-CdR) or trichostatin A (TSA) alone could partially reverse 3OST2 methylation, and their combination resulted in complete reversal. UHRF1 and histone H3R8me2s were both enriched on the hypermethylated 3OST2 promoter, but H3R8me2a was not. After 5-Aza-CdR or TSA treatment, the UHRF1 and H3R8me2s enrichment was decreased, while H3R8me2a enrichment increased. We demonstrated that 3OST2 methylation may play a critical role in the earliest steps of hepatocarcinogenesis and is directly regulated by UHRF1. Furthermore, H3R8me2s acted as a repressive mark, while H3R8me2a was correlated with 3OST2 transcriptional activity.