| Objective To identify the abnormal methylated biomarkers in hepatocellular carcinoma and explore their potential clinical significance.Methods (1) The methylation levels of acid phosphatase locus 1 (ACP1), bone morphogenetic protein 4 (BMP4) and testis-specific Y-like protein 5 (TSPYL5) were analyzed in 188 HCC tissues,163 matched adjacent non-tumor tissues,29 normal liver tissues and 34 plasma samples using a method of methylation-sensitive restriction enzyme based quantitative PCR (MSRE-qPCR), and their associations with clinicopathological features and prognosis were evaluated. Moreover, bisulfite genomic sequencing (BGS) was used to validate the result of MSRE-qPCR in 30 paired HCC and adjacent non-tumor tissues. (2) We analyzed the methylation level of tripartite motif containing 58 (TRIMS8) in 181 HCC tissues,172 matched adjacent non-tumor tissues and 13 normal liver tissues using MSRE-qPCR and BGS. Furthermore, the mRNA expression level of TRIM58 was measured in 46 paired HCC and adjacent non-tumor tissues by quantitative real-time PCR. Meanwhile, the relationships between TRIM58 methylation and mRNA expression, the clinicopathological features, as well as prognostic value were evaluated. (3) Genome-wide DNA methylation analysis was conducted in 3 paired hepatocellular carcinoma and adjacent non-tumor tissues using reduced representation bisulfite sequencing (RRBS). Illumina MiSeq sequencing-based bisulfite sequencing PCR (Miseq-BSP) and MSRE-qPCR were used to validate the data from RRBS with a large sample size. The correlations between DNA methylation and clinicopathological features and prognosis were evaluated.Results (1) Compared with adjacent non-tumor tissues and normal liver tissues, the methylation levels of ACPI, BMP4 and TSPYLS were significantly increased in HCC tissues (All p<0.0001). For plasma samples, the methylation level of TSPYL5 was significant higer in HCC patients than controls (p= 0.001) even with low methylation level. The methylation of each individual gene could distinguish HCC tissues well from adjacent non-tumor tissues with the area under the receiver operating characteristic curves (AUC) of 0.753,0.785 and 0.917, respectively. Furthermore, a higher methylation of BMP4 was statistically associated with worse disease-free survival (p= 0.006) and might be an independent unfavorable factor for disease-free survival by univariate and multivariate analysis (p= 0.011, HR= 3.431,95% CI: 1.333-8.833). (2) TRIM58 methylation was significantly higher in HCC tissues compared with adjacent non-tumor tissues and normal liver tissues (both p< 0.0001). Hypermethylation of TRIM58 was specific in HCC tissues (28.18%,51/181) and tend to correlate with unfavorable disease-free survival (p=0.047). Moreover, the mRNA expression of TRIM58 was significantly decreased in HCC tissues compared with adjacent non-tumor tissues (p< 0.0001), and showed a negative association with DNA methylation (p= 0.015, rs=-0.260). (3) RRBS developed 3298,5809 and 18402 differentially methylated regions (DMR) in each paired HCC and adjacent non-tumor tissues, respectively. And there were 975 common DMRs among 3 pairs of HCC and adjacent non-tumor tissues, corresponding to 396 annotated genes. Miseq-BSP analyzed the methylation of ARHGAP6 (Rho GTPase activating protein 6), BEND4 (BEN domain containing 4), CAMK2B (calcium/calmodulin dependent protein kinase II beta), DGKE (diacylglycerol kinase epsilon), PTH2R (parathyroid hormone 2 receptor), VIM (vimentin) in 33 HCC and paired adjacent non-tumor tissues, as well as 6 normal liver tissues. The methylation levels of those genes in HCC tissues were significant higher than adjacent non-tumor tissues (p< 0.0001), which were consistent with RRBS. Spearman’s rank order correlation coefficient was calculated to further analyzed associations between RRBS and Miseq-BSP. And they showed a significantly positive correlation for each gene (rs> 0, p< 0.0001), especially for ARHGAP6 (rs= 0.851, p= 4.67×10-28). ARHGAP6 methylation was further validated in 211 HCC tissues,193 matched adjacent non-tumor tissues and 20 normal liver tissues, and found that its methylation was significant increased in HCC compared with adjacent non-tumor tissues (p< 0.0001) and normal liver tissues (p< 0.0001). Moreover, the methylation level of ARHGAP6 had a significant positive correlation with serum AFP level (p= 0.001, rs= 0.235).Conclusions (1) Hypermethylation of ACPI, BMP4 and TSPYL5 are common events in HCC and could be used as potentially detectable biomarkers in HCC. Moreover, BMP4 could be potentially served as a methylated biomarker to predict recurrence and metastasis after hepatectomy for HCC patients. (2) TRIM58 methylation is a common event in HCC and may contribute to downregulation of its mRNA expression. Furthermore, hypermethylation of TRIM58 tend to associate with worse DFS after hepatectomy. (3) RRBS was used to analyze the genome-wide methylation of HCC at single nucleotide resolution, and which enriched the DNA methylation map of HCC. Several novel differential methylated genes were identified, and were validated in a large sample size. |
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