Association of 5-methylcytosine and 5-hydroxymethylcytosine with mitochondrial DNA content and clinical and biochemical parameters in hepatocellular carcinoma

Abstract

Increasing epidemiological evidence has indicated that inherited variations of mitochondrial DNA (mtDNA) copy number affect the genetic susceptibility of many malignancies in a tumour-specific manner and that DNA methylation also plays an important role in controlling gene expression during the differentiation and development of hepatocellular carcinoma (HCC). Our previous study demonstrated that HCC tissues showed a lower 5-hydroxymethylcytosine (5-hmC) content when compared to tumour-adjacent tissues, but the relationship among 5-hmC, 5-methylcytosine (5-mC) and mtDNA content in HCC patients is still unknown. This study aimed to clarify the correlation among mtDNA content, 5-mC and 5-hmC by quantitative real-time PCR and liquid chromatography tandem mass spectrometry analysis. We demonstrated that 5-hmC correlated with tumour size [odds ratio (OR) 0.847, 95% confidence interval (CI) 0.746-0.962, P = 0.011], and HCC patients with a tumour size ≥ 5.0 cm showed a lower 5-hmC content and higher levels of fasting plasma aspartate aminotransferase, the ratio of alanine aminotransferase to aspartate aminotransferase, γ-glutamyltransferase, alpha-fetoprotein than those with a tumour size <5 cm (all P<0.05). We further revealed that the mtDNA content of HCC tumour tissues was 225.97(105.42, 430.54) [median (25th Percentile, 75th Percentile)] and was negatively correlated with 5-mC content (P = 0.035), but not 5-hmC content, in genomic DNA from HCC tumour tissues.

Figure 1. Comparisons of 5-hmC and clinico-pathological parameters in HCC based on tumour size.

Figure 2. Comparisons of 5-mC and clinico-pathological parameters in HCC based on liver inflammation.

0 = the group without HBV or HCV infection (n = 20), 1 = the mild hepatitis group (n = 33), 2 = the moderate hepatitis group (n = 31), 3 = the severe hepatitis group (n = 26), 4 = the liver failure group (n = 34). Bar charts and Boxplots indicate the results of One-way ANOVA and K-Independent non-parametric analysis, respectively. □: Group-0 vs. Group-3, ▴: Group-0 vs. Group-4, ▪: Group-1 vs. Group-2, #: Group-1 vs. Group-3, ∇: Group-1 vs. Group-4, ▾: Group-2 vs. Group-3, ○: Group-2 vs. Group-4, •: Group-3 vs. Group-4.