Methylation Profiling of Multiple Tumor Suppressor Genes in Hepatocellular Carcinoma and the Epigenetic Mechanism of 3OST2 Regulation - PubMed (original) (raw)

. 2015 Jun 25;6(8):740-9.

doi: 10.7150/jca.11691. eCollection 2015.

Affiliations

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

Haiyan Chen et al. J Cancer. 2015.

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.

Keywords: DNA methylation; Gene expression; Hepatocellular carcinoma; Histone modification; Tumor suppressor gene.

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Conflict of interest statement

Conflict of Interest: There are no conflicts of interest.

Figures

Figure 1

Figure 1

Representative samples (n=3) of the methylation status of ten umor suppressor genes (TSGs) in 48 hepatocellular carcinomas (HCCs) and matched corresponding noncancerous tissues. The methylation-specific PCR (MSP) products in the M lanes indicate the presence of methylated alleles, and those in the U lanes indicate the presence of unmethylated allele; N, non-tumor; T, tumor.

Figure 2

Figure 2

(A) The methylation status of the 3OST2 promoter in normal liver cells and in HCC cells with or without 5-Aza-CdR and/or TSA treatment. The 3OST2 promoter was not methylated in normal liver cells and completely methylated (only methylated alleles were present) in HCC cells. This was partially reversed by 5-Aza-CdR or TSA treatment (both unmethylated and methylated alleles were present). When 5-Aza-CdR was combined with TSA, the promoter was completely reversed (only unmethylated alleles were present). (B) Real-time reverse transcription PCR (qRT-PCR) analysis of 3OST2 mRNA expression in HCC cells. Expression was normalized using β-actin as an internal control.

Figure 3

Figure 3

(A) Immunohistochemical analysis of UHRF1 protein expression in HCC cells with or without 5-Aza-CdR and/or TSA treatment. (a) Untreated HCC cells; (b) after 5-Aza-CdR treatment; (c) after TSA treatment; (d) after 5-Aza-CdR and TSA combined treatment; (e) Untreated normal liver cell line QSG-7701 cells. (B) qRT-PCR analysis of UHRF1 mRNA expression (C, D) Western blot analysis of UHRF1 protein expression; the ratio of UHRF1 to β-actin is shown on the y-axis. (E) Chromatin immunoprecipitation (ChIP)-quantitative real time PCR (qPCR) assay for UHRF1 on the 3OST2 promoter. Shown is the enrichment, relative to the input obtained in each ChIP sample.

Figure 4

Figure 4

(A) Western blot analysis of three methylation states of H3R8 (H3R8me1, H3R8me2s and H3R8me2a) in normal liver cells and HCC cells with or without 5-Aza-CdR and/or TSA treatment. (B-E) The ratio of H3R8me1/H3R8me2s/H3R8me2a to β-actin is shown on the y-axis. Note that H3R8me2a was not present in the HCC cells.

Figure 5

Figure 5

ChIP-qPCR assays for H3R8me2s and H3R8me2a on the 3OST2 promoter in normal liver cells and HCC cells with or without 5-Aza-CdR and/or TSA treatment. Cross-linked chromatin from HCC cells treated or untreated with 5-Aza-CdR and/or TSA underwent immunoprecipitation using control IgG, anti-UHRF1, anti-H3R8me2s or anti-H3R8me2a antibodies; the immunoprecipitated DNA was analyzed by qPCR using primers specific for 3OST2. Shown is the enrichment, relative to the input obtained in each ChIP samples.

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