Abstract: Objective To clarify the application value of 5-hydroxymethylcytosine (5hmC) in evaluating the progression risk of chronic hepatitis B (CHB) to hepatocellular carcinoma (HCC) based on differential analysis. Methods Eighty-four CHB patients without HCC progression were included in the control group (CG), and 96 CHB patients with HCC development were included in the research group (RG). Two-thirds of the samples were used in the training set and one-third of the samples were used in the validation set to detect the level of 5hmC in both groups based on a modified nano-hmC-Seal technique. The expression levels of 5hmC-related genes TET2 and TET3 were quantified by qPCR method, and the correlation between TET3 and 5hmC was analyzed by Pearson′s correlation coefficients method. Receiver operating characteristic (ROC) curves were drawn to evaluate the application value of the TET3-based 5hmC prediction model in the early diagnosis of HCC. Results The expression of 5hmC in patients of RG group was lower than that in CG group, no matter in the training set or in the validation set (training set: 0.73±0.31 vs.0.98±0.31, validation set: 1.01±0.34 vs.2.98±1.25). 5hmC was significantly enriched in the region between the transcription initiation site and the transcription ending site but was depleted in the flanking region. 5hmC-related genes TET2 and TET3 were significantly down-regulated in HCC patients, both in the training set and in the validation set (training set: TET2 0.72±0.26 vs.1.04±0.36, TET3 0.73±0.34 vs.0.96±0.34, validation set: TET2 1.89±0.28 vs.3.25±1.25, TET3 0.71±0.22 vs.1.04±0.27). In both the training and validation sets, TET3 showed a positive association with 5hmC. ROC analysis results showed that the 5hmC prediction model could be used to predict the progression of CHB to HCC (training set: AUC=0.81, 0.729~0.893; validation set: AUC=0.84, 0.739~0.936). Conclusion TET3 expression based on 5hmC sequencing is a landmark molecule for evaluating the progression of HCC in CHB patients, which is worthy of further study and promotion.

Key words: Hepatitis B virus, Chronic hepatitis B, Hepatocellular carcinoma, 5hmC modification