Evaluation of HAAH/humbug quantitative detection in the diagnosis of hepatocellular carcinoma

Human aspartyl-(asparaginyl)-β-hydroxylase (HAAH) is a type 2 transmembrane protein and an α-ketoglutarate- dependent dioxygenase that can stereospecifically catalyze the post-translational hydroxylation reaction of β-carbon atoms of aspartic acid and asparagine residues present in epidermal growth factor-like domains of certain specific proteins. Humbug is a truncated isoform of aspartyl (asparaginyl) β-hydroxylase that lacks the catalytic domain. A series of reports demonstrated that overexpression of HAAH/humbug was identified in hepatocellular carcinoma (HCC) and various tumor tissues. However, the prognostic value of HAAH/humbug expression in HCC remains unclear. The purpose of this study was to investigate the expression of the HAAH/humbug gene at the mRNA and protein levels in HCC and to assess the overexpression of HAAH/humbug as a diagnostic and prognostic marker in HCC. HAAH/humbug mRNA levels were measured in 120 HCCs and 40 paired non- tumor liver tissues by molecular beacon (MB) quantitative RT-PCR. Immunohistochemical staining was used to detect the protein level of the HAAH/humbug in the same speci- mens. ROC analysis was performed based on the expression levels of the HAAH/humbug gene in 120 cases of HCC tissues and 40 cases of adjacent non-tumor liver tissues. The results showed that 117 (97.5%) of the 120 frozen sections of patients with HCCs had HAAH/humbug-positive immunoreactivity, whereas the 40 adjacent non-tumor liver tissues exhibited no staining. Higher levels of HAAH/humbug mRNA were found in 114 (95%) of the 120 HCC tissues relative to the adjacent cancer-free tissue. ROC curve analysis exhibited that the sensitivity was 90.1%, specificity was 97.6% and ROC AUC was 0.986. The specific value of HAAH/β-actin abundance used as a cut-off point was 0.315, while the gene copy number (7.35 copies/µl) was used a as cut-off point, with sensitivity being 99.2%, specificity 96.7% and the ROC AUC used 0.990. No statistically significant difference was observed for these two factors. HAAH/humbug expression levels were upregu- lated in almost all the HCC tissues when compared to the adjacent cancer-free tissue, irrespective of the cut-off point used. Results of the present study suggested that HAAH/ humbug is a potential diagnostic and prognostic biomarker for the treatment of HCC.