Feng-Kai Sun

Methylation of serum insulin‐like growth factor‐binding protein 7 promoter in hepatitis B virus‐associated hepatocellular carcinoma

Methylation of gene promoter CpG islands is an important early event in hepatocellular carcinoma (HCC), and detection of cell‐free tumor‐specific DNA methylation is becoming a useful noninvasive method for HCC. This study was aimed at determining the diagnostic value of serum insulin‐like growth factor‐binding protein 7 (IGFBP7) promoter methylation in hepatitis B virus‐associated HCC. A total of 217 subjects, including 136 HCC patients, 46 patients with chronic hepatitis B (CHB), and 35 healthy controls (HCs), were included. The methylation status of the serum IGFBP7 gene promoter was determined using methylation‐specific PCR. The frequency of serum IGFBP7 promoter methylation in HCC patients (89/136, 65%) was significantly higher than that in CHB patients (8/46, 17%; X2 = 31.883, P < 0.001) and HCs (5/35, 14%; X2 = 29.429, P < 0.001). Moreover, elevated IGFBP7 methylation frequency was also observed in HCC patients with vascular invasion compared with those without vascular invasion (84 versus 60%, X2 = 6.633, P = 0.010). The sensitivities of serum IGFBP7 methylation and alpha‐fetoprotein (AFP) in detecting HCC were 65 and 57%, respectively. Of note, the combination of IGFBP7 methylation and AFP showed 85% for sensitivity. These results suggest that methylation of the serum IGFBP7 gene promoter may serve as a useful noninvasive biomarker for HCC diagnosis.

Elevated expression of tumour necrosis factor‐α‐induced protein 8 (TNFAIP8)‐like 2 mRNA in peripheral blood mononuclear cells is associated with disease progression of acute‐on‐chronic hepatitis B liver failure

Aberrant immunity response contributes to the pathogenesis of acute‐on‐chronic hepatitis B liver failure. Tumour necrosis factor‐α‐induced protein‐8 like‐2 (TIPE2) is a recently identified molecular to maintain immune homoeostasis, but its role in acute‐on‐chronic hepatitis B liver failure (ACHBLF) is unknown. We detected TIPE2 mRNA levels in peripheral blood mononuclear cells (PBMCs) from 56 patients with ACHBLF, 60 chronic hepatitis B patients, 24 healthy controls and analysed its role in disease severity and prognosis. TIPE2 mRNA expression in patients with ACHBLF was higher than that of patients with chronic infection or healthy controls. In patients with ACHBLF, TIPE2 mRNA level was positively correlated with serum total bilirubin, international normalized ratio and model for end‐stage liver disease scores. Furthermore, the level of TIPE2 mRNA was significantly higher in nonsurvivors than survivors in patients with ACHBLF. The mRNA level of TIPE2 gradually decreased week by week in survivors accompanied by recovery from patients with ACHBLF, while its expression sustained at high levels in nonsurvivors. TIPE2 mRNA level after lipopolysaccharide (LPS) stimulation ex vivo in patients with ACHBLF was higher compared with controls and patients with chronic infection. Meanwhile, cytokines ex vivo secreted were measured as a marker of immune activation. After LPS stimulation, interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α mRNA expression were reduced in patients with ACHBLF, and a significantly negative correlation was found between TIPE2 and TNF‐α mRNA levels. In conclusion, our results identified the potential role of TIPE2 in predicting disease progression and prognosis in patients with ACHBLF by negative regulating of cell‐mediated immunity.

Methylation of tissue factor pathway inhibitor 2 as a prognostic biomarker for hepatocellular carcinoma after hepatectomy.

Methylation of tissue factor pathway inhibitor 2 (TFPI2) gene has been detected in hepatocellular carcinoma (HCC). However, the clinicopathologcial significance and prognostic value of TFPI2 methylation in HCC remains largely unknown. This study aimed to investigate the prognostic value of TFPI2 methylation in HCC after hepatectomy.

Aberrant GSTP1 promoter methylation predicts short-term prognosis in acute-on-chronic hepatitis B liver failure

Glutathione‐S‐transferase P1 (GSTP1) methylation has been demonstrated to be associated with oxidative stress induced liver damage in acute‐on‐chronic hepatitis B liver failure (ACHBLF).

Exportin 4 gene expression and DNA promoter methylation status in chronic hepatitis B virus infection.

Exportin 4 (XPO4) is a novel identified candidate tumour‐suppressor gene involved in the pathogenesis of hepatocellular carcinoma (HCC). This study was aimed to determine the clinical features of XPO4 mRNA expression and promoter methylation status in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis B virus (HBV) infection. PBMCs were isolated from 44 HCC, 38 liver cirrhosis (LC), 34 chronic hepatitis B (CHB) patients and 17 healthy controls (HCs). The mRNA level and promoter methylation status of XPO4 were determined by quantitative real‐time RT‐PCR and methylation‐specific PCR, respectively. XPO4 mRNA level of HCC patients was significantly lower compared with LC and CHB patients as well as HCs (all P < 0.01, respectively), and significant differences of the XPO4 mRNA level were found in LC and CHB group than in HCs (LC vs HCs, P < 0.01; CHB vs HCs, P < 0.05). Methylation rate of XPO4 promoter was significantly increased in patients with HCC than in patients with CHB and HCs (both P < 0.05). DNA methylation pattern was responsible for the suppression of XPO4 transcription in the progression of HBV infection (P = 0.000). Furthermore, AFP level was significantly higher in HCC patients with XPO4 methylation than in those without methylation ((8702 ± 15635) μm vs (1052 ± 5370) μm, P < 0.05). In conclusion, transcription of XPO4 gene was gradually decreased and methylation rate of XPO4 promoter was increased with the progression of HBV infection. Methylation status of XPO4 in PBMCs tended to be a noninvasive biomarker to predict HCC and the progression of HBV infection.

Peripheral Type I Interferon Receptor Correlated with Oxidative Stress in Chronic Hepatitis B Virus Infection

Type I interferon receptor (IFNAR) has been involved in the progression of chronic hepatitis B (CHB). Oxidative stress is also associated with hepatitis B virus (HBV) infection and might contribute to the structure and function of protein synthesis including the IFNAR family. This study was aimed to determine the possible associations between oxidative stress and peripheral IFNAR expression in chronic HBV infection. Fifty-four CHB patients and 31 liver cirrhosis (LC) patients were consecutively collected, as well as 11 healthy subjects as controls. Expression levels of IFNAR1 and IFNAR2 in peripheral blood lymphocytes and monocytes were measured by flow cytometry. IFNAR1 and IFNAR2c mRNA were detected by real-time reverse transcription-polymerase chain reaction. Levels of plasma-soluble IFNAR and oxidative stress parameters, including xanthine oxidase (XOD), malondialdehyde (MDA), glutathione (GSH), glutathione S-transferase (GST), and glutathione peroxidase (GSH-Px) were detected by enzyme linked immunosorbent assay (ELISA). The frequencies of IFNAR1 and IFNAR2 in lymphocytes and monocytes were significantly increased in CHB and LC patients than in healthy controls. Expression levels of IFNAR1 and IFNAR2c mRNA and plasma-soluble IFNAR level in CHB and LC patients were upregulated compared with healthy controls. Mean fluorescence intensity (MFI) of IFNAR2 in monocytes of CHB patients was higher than that in LC patients. Levels of plasma XOD, MDA, and GST were significantly increased in CHB and LC patients compared with healthy controls. Meanwhile, GSH and GSH-Px in CHB and LC patients were decreased than that in healthy controls. Furthermore, plasma MDA, GSH, and GST levels in CHB patients were higher than that in LC patients. In CHB patients, plasma GST level was negatively correlated with MFI of IFNAR2 in lymphocytes. Our results suggested that oxidative stress play an important role in the regulation of IFNAR in chronic HBV infection.

Aberrant DNA methylation of G‐protein‐coupled bile acid receptor Gpbar1 predicts prognosis of acute‐on‐chronic hepatitis B liver failure

The G‐protein‐coupled bile acid receptor Gpbar1 (TGR5) has been demonstrated to be able to negatively regulate hepatic inflammatory response. In this study, we aimed to determine the methylation status of TGR5 promoter in patients with acute‐on‐chronic hepatitis B liver failure (ACHBLF) and its predictive value for prognosis. We enrolled 76 consecutive ACHBLF patients, 80 chronic hepatitis B (CHB) patients and 30 healthy controls (HCs). Methylation status of TGR5 promoter in peripheral mononuclear cell (PBMC) was detected by methylation‐specific polymerase chain reaction (MSP). The mRNA level of TGR5 was determined by quantitative real‐time polymerase chain reaction (RT‐qPCR). We found that the frequency of TGR5 promoter methylation was significantly higher in ACHBLF (35/76, 46.05%) than CHB patients (5/80, 6.25%; χ2 = 32.38, P < 0.01) and HCs (1/30, 3.33%; χ2 = 17.50, P < 0.01). TGR5 mRNA level was significantly lower (Z = −9.12, P < 0.01) in participants with aberrant methylation than those without. TGR5 methylation showed a sensitivity of 46.05% (35/76), specificity of 93.75% (75/80), positive predictive value (PPV) of 87.5% (35/40) and negative predictive value (NPV) of 64.66% (75/116) in discriminating ACHBLF from CHB patients. ACHBLF patients with methylated TGR5 showed significantly poor survival than those without (P < 0.01). When used to predict 3‐month mortality of ACHBLF, TGR5 methylation [area under the receiver operating characteristic curve (AUC) = 0.75] performed significantly better than model for end‐stage liver diseases (MELD) score (AUC = 0.65; P < 0.05). Therefore, our study demonstrated that aberrant TGR5 promoter methylation occurred in ACHBLF and might be a potential prognostic marker for the disease.

Methylation of suppressor of cytokine signalling 1 gene promoter is associated with acute‐on‐chronic hepatitis B liver failure

Suppressor of cytokine signalling 1 (SOCS1) was demonstrated to play an important negative role in fulminant hepatitis and might be involved in acute‐on‐chronic hepatitis B liver failure (ACHBLF). This study was therefore to identify the potential role of SOCS1 and its promoter methylation pattern in ACHBLF patients. Sixty ACHBLF patients, 60 chronic hepatitis B (CHB) patients and 30 healthy controls were investigated in this study. We found that expression of SOCS1 mRNA in CHB and ACHBLF patients was significantly higher than that in healthy controls. The serum level of IL‐6, IFN‐γ and TNF‐α was significantly higher in ACHBLF than CHB. Increased serum level of IL‐6, IFN‐γ and TNF‐α was correlated with total bilirubin, ALT, PTA and MELD scores in ACHBLF. The degree of methylation of the SOCS1 in ACHBLF patients (35.0%, 21/60) was significantly higher than that in CHB patients (16.7%, 10/60). Furthermore, methylated group showed lower level of SOCS1, and higher MELD scores and mortality rate when compared with unmethylated group of ACHBLF. These results suggested that SOCS1 might contribute to immune‐related liver damage in ACHBLF, and its aberrant methylation may be a key event for the prognosis of ACHBLF.

A model to predict antiviral treatment in HBeAg negative chronic hepatitis B with alanine aminotransferase ≤2 upper limit of normal

Liver histological assessment is essential for predicting antiviral therapy in HBeAg negative chronic hepatitis B (CHB) patients with serum alanine aminotransferase (ALT) ≤2 upper limit of normal (ULN). The aim was to establish a model to predict antiviral treatment for those patients without liver biopsy.

High promoter methylation levels of glutathione-S-transferase M3 predict poor prognosis of acute-on-chronic hepatitis B liver failure

This study aimed to evaluate the prognostic value of glutathione‐S‐transferase M3 (GSTM3) gene promoter methylation in patients with acute‐on‐chronic hepatitis B liver failure (ACHBLF).