Hypomethylation of glycine dehydrogenase promoter in peripheral blood mononuclear cells is a new diagnostic marker of hepatitis B virus-associated hepatocellular carcinoma.

BACKGROUND: Glycine dehydrogenase (GLDC) plays an important role in the initiation and proliferation of several human cancers. In this study, we aimed to detect the methylation status of GLDC promoter and its diagnostic value for hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC). METHODS: We enrolled 197 patients, 111 with HBV-HCC, 51 with chronic hepatitis B (CHB), and 35 healthy controls (HCs). The methylation status of GLDC promoter in peripheral mononuclear cells (PBMCs) was identified by methylation specific polymerase chain reaction (MSP). The mRNA expression was examined using real-time quantitative polymerase chain reaction (qPCR). RESULTS: The methylation frequency of the GLDC promoter was significantly lower in HBV-HCC patients (27.0%) compared to that in CHB patients (68.6%) and HCs (74.3%) (P < 0.001). The methylated group had lower alanine aminotransferase level (P = 0.035) and lower rates of tumor node metastasis (TNM) III/IV (P = 0.043) and T3/T4 (P = 0.026). TNM stage was identified to be an independent factor for GLDC promoter methylation. GLDC mRNA levels in CHB patients and HCs were significantly lower than those in HBV-HCC patients (P = 0.022 and P < 0.001, respectively). GLDC mRNA levels were significantly higher in HBV-HCC patients with unmethylated GLDC promoters than those with methylated GLDC promoters (P = 0.003). The diagnostic accuracy of alpha-fetoprotein (AFP) combined with GLDC promoter methylation for HBV-HCC was improved compared with that of AFP alone (AUC: 0.782 vs. 0.630, P < 0.001). In addition, GLDC promoter methylation was an independent predictor for overall survival of HBV-HCC patients (P = 0.038). CONCLUSIONS: The methylation frequency of GLDC promoter was lower in PBMCs from HBV-HCC patients than that from patients with CHB and HCs. The combination of AFP and GLDC promoter hypomethylation significantly improved the diagnostic accuracy of HBV-HCC.

F-box protein 43 promoter methylation as a novel biomarker for hepatitis B virus-associated hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) has a high prevalence and poor prognosis worldwide. Therefore, it is urgent to find effective and timely diagnostic markers. The objective of this study was to evaluate the diagnostic value of F-box protein 43 promoter methylation in peripheral blood mononuclear cells (PBMCs) for HCC. Method: A total of 247 participants were included in this study, comprising individuals with 123 hepatitis B virus-associated HCC, 79 chronic hepatitis B, and 45 healthy controls. F-box protein 43 methylation and mRNA levels in PBMCs were detected by MethyLight and quantitative real-time PCR. Result: F-box protein 43 promoter methylation levels were significantly lower in HCC PBMCs than the chronic hepatitis B (P < 0.001) and healthy control PBMCs (P < 0.001). Relative mRNA expression levels of F-box protein 43 in HCC PBMCs were significantly higher than those in chronic hepatitis B (P < 0.001) and healthy control PBMCs (P < 0.001). Receiver operating characteristic analysis of F-box protein 43 promoter methylation levels yielded an area under curve (AUC) of 0.793 with 76.42% sensitivity and 68.35% specificity when differentiating HCC from chronic hepatitis. These values for the F-box protein 43 promoter methylation level were superior to those of the alpha-fetoprotein serum (AFP) level (AUC: 0.780, sensitivity: 47.97%, and specificity: 96.20%), with increments in values for the combination of F-box protein 43 promoter methylation AFP levels (AUC: 0.888, sensitivity: 76.42%, and specificity: 86.08%). Conclusion: Hypomethylation of the F-box protein 43 promoter in PBMCs is a promising biochemical marker for HBV-associated HCC.

Hypomethylation of Tumor necrosis factor-like cytokine 1A(TL1A) and its decoy receptor 3 expressive level increase has diagnostic value in HBV-associated cirrhosis.

For patients with cirrhosis, early diagnosis is the key to delaying the development of liver fibrosis and improving prognosis. This study aimed to investigate the clinical significance of TL1A, which is a susceptibility gene for hepatic fibrosis, and DR3 in the development of cirrhosis and fibrosis. We analyzed the expression of TL1A, DR3, and other inflammatory cytokines associated with liver fibrosis in serum and PBMCs in 200 patients.TL1A methylation level was lower in patients with HBV-associated LC than in the other groups. In addition, the mRNA level and serum of TL1A and DR3 expression levels were found to increase in the LC. Hypomethylation of the TL1A promoter is present in HBV-associated LC, and TL1A and DR3 are highly expressed in HBV-associated cirrhosis. These results indicate that TL1A and DR3 may play an important role in the pathogenesis of LC and TL1A methylation levels may serve as a noninvasive biomarker for early diagnosis and progression of LC.

Assessing causal relationships between sarcopenia and nonalcoholic fatty liver disease: A bidirectional Mendelian randomization study.

Background and aims: Sarcopenia has been demonstrated to be closely associated with nonalcoholic fatty liver disease (NAFLD). However, whether there are causal relationships between sarcopenia and NAFLD remains undetermined. Here, we aim to address the question using a two-sample bidirectional Mendelian randomization (MR) analysis approach. Methods: We performed a two-sample bidirectional MR study using summary-level data from genome-wide association studies (GWAS) of the whole body lean mass (n = 38,292), appendicular (arms and legs) lean mass (n = 28,330), and NAFLD (1,483 biopsy-proven NAFLD cases and 17,781 controls). We first conducted MR analysis with five single nucleotide polymorphisms (SNPs) as genetic instruments for whole body lean mass and three SNPs as instruments for appendicular lean mass to estimate the causal effect of genetically predicted sarcopenia on the risk of NAFLD using the inverse-variance weighted (IVW) method. Then we performed reverse MR analysis with four SNPs as instruments to examine the causality of genetically predicted NAFLD with whole body lean mass and appendicular lean mass. Further sensitivity analysis was conducted to testify the reliability of the MR results. Results: Genetic predisposition to decreased whole body lean mass was not associated with NAFLD [IVW-random effects, odds ratio (OR) = 1.054, 95%CI: 0.750-1.482, P = 0.761]. Similar results were observed using genetic instruments of appendicular lean mass (IVW-random effects, OR = 0.888, 95%CI: 0.386-2.042, P = 0.780). Reverse MR analysis revealed that genetically predicted NAFLD using four genetic instruments was not associated with whole body lean mass (IVW, ß = -0.068, 95%CI: -0.179 to 0.043, P = 0.229) and appendicular lean mass (IVW, ß = -0.020, 95%CI: -0.092 to 0.051, P = 0.574). MR analyses using other methods and sensitivity analysis showed consistent results. Conclusion: These results suggested no causal relationships between sarcopenia and NAFLD, indicating that sarcopenia may not be directly involved in the pathogenesis of NAFLD and vice versa.

Acute hepatitis of unknown origin in children: A combination of factors.

On 5 April 2022, the World Health Organization was notified of 10 cases of severe acute hepatitis of unknown etiology in children under 10 years of age in the United Kingdom. Although the exact cause of a proportion of pediatric acute hepatitis and acute liver failure cases was unclear, the above event has caused widespread concern worldwide. As of 14 September 2022, approximately 1,296 probable cases of acute hepatitis of unknown etiology have been reported from 37 countries/regions, of which approximately 55 required or received liver transplantation and 29 died. Although the etiology of acute hepatitis of unknown origin in children remains unclear, many hypotheses have been proposed about the disease. Instead of individual factors such as "adenovirus infection," "SARS-CoV-2 related," and "Adeno-associated virus 2 with helper virus coinfection," it is more likely due to a combination of factors. Accordingly, there is an urgent need for more data and research to clarify the disease etiology. This review aims to provide a historical perspective of acute hepatitis of unknown etiology in children in the past decades and summarize the current hypothesis and evidence on this emerging disease.

IL-6 Promoter Hypomethylation Acts As a Diagnostic Biomarker in Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Background: New biomarkers are needed to detect hepatocellular carcinoma at an earlier stage and to individualize treatment strategies. IL-6 has been proven to be associated with liver cancer in numerous studies. Aim: To evaluate the value of the IL-6 promoter methylation level as a noninvasive biomarker for the diagnosis of liver cancer. Methods: A retrospective analysis of 165 patients with HBV-associated hepatocellular carcinoma (HCC), 198 patients with chronic hepatitis B (CHB) and 31 healthy controls were involved. The methylight was detected the methylation level of the IL-6 promoter in peripheral blood mononuclear cells (PBMCs), clinical and laboratory parameters were obtained. Results: IL-6 promoter methylation levels were significantly lower in patients with HCC (median 53.59%, interquartile range 52.01-54.75%) than in those with CHB (median 56.05%, interquartile range 54.65-57.67%; P<0.001). The level of IL-6 mRNA in patients with HCC (median 0.371, interquartile range 0.173-0.671) was significantly higher than that in patients with CHB (median 0.203, interquartile range 0.108-0.354; P<0.001) and HCs (median 0.189, interquartile range 0.140-0.262; P=0.001). Meanwhile, the PMR value of IL-6 was notably negatively correlated with the mRNA expression level (Spearman's R=-0.201, P<0.001). The IL-6 PMR value of HCC patients in age (Spearman's R=0.193, P=0.026) and TBIL (Spearman's R=0.186, P=0.032) were very weak correlated. At the same time, the level of IL-6 promoter methylation was also an independent factor in the development of liver cancer. When the IL-6 promoter methylation level was used to diagnose HCC, its detective value was superior to AFP [area under the receiver operating characteristic curve (AUC) 0.773 vs. 0.686, P=0.027], And the combined use of AFP and IL-6 methylation level can improve the area under the receiver operating characteristic curve (p=0.011). Conclusion: IL-6 promoter hypomethylation is present in hepatocellular carcinoma, and it may be used as a noninvasive biomarker to detect early liver cancer.

Prognostic potential of the small GTPase Ran and its methylation in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignant tumor with high mortality. The prognostic significance of Ran, a member of Ras superfamily, remains unclear in HCC patients. METHODS: Based on The Cancer Genome Atlas (TCGA) database and Tumor Immune Estimation Resource (TIMER), we analyzed the correlations among Ran expression, promoter methylation and immune cell infiltration. We also investigated the Ran expression levels in HCC tissues and normal tissues by using quantitative real-time PCR. RESULTS: Ran mRNA expression was significantly increased in HCC tissues compared with the normal tissues (P < 0.001). Time-dependent receiver operating characteristic (ROC) curves showed that Ran expression had predictive value of the 1-, 3- and 5-year overall survival for HCC patients, and the areas under the curves (AUC) were 0.747, 0.634 and 0.704, respectively. Cox regression analysis showed that Ran expression was an independent prognostic factor for HCC patients (HR = 1.492, 95% CI: 1.129-1.971, P = 0.005). We also found a negative relationship between Ran mRNA expression and its promoter methylation (r = -0.36, P < 0.001). High Ran expression and promoter hypomethylation predicted worse overall survival and progression-free survival (P < 0.05) and were involved in the progression of HCC. Ran expression exhibited significant correlations with immune infiltrates and prognostic immune-related genes. CONCLUSIONS: The present study provides further insight into the prognosis of HCC, and Ran could serve as a biomarker for predicting the survival of HCC patients.

Noncanonical Wnt5a/JNK Signaling Contributes to the Development of D-Gal/LPS-Induced Acute Liver Failure.

Acute liver failure (ALF) is a deadly clinical disorder with few effective treatments and unclear pathogenesis. In our previous study, we demonstrated that aberrant Wnt5a expression was involved in acute-on-chronic liver failure. However, the role of Wnt5a in ALF is unknown. We investigated the expression of Wnt5a and its downstream c-Jun N-terminal kinase (JNK) signaling in a mouse model of ALF established by coinjection of D-galactosamine (D-Gal) and lipopolysaccharide (LPS) in C57BL/6 mice. We also investigated the role of Box5, a Wnt5a antagonist, in vivo. Moreover, the effect of Wnt5a/JNK signaling on downstream inflammatory cytokine expression, phagocytosis, and migration in THP-1 macrophages was studied in vitro. Aberrant Wnt5a expression and JNK activation were detected in D-Gal/LPS-induced ALF mice. Box5 pretreatment reversed JNK activation and eventually decreased the mortality rate of D-Gal/LPS-treated mice, with reduced hepatic necrosis and apoptosis, serum ALT and AST levels, and liver inflammatory cytokine expression, although the latter was not significant. We further demonstrated that recombinant Wnt5a (rWnt5a)-induced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expression and increased THP-1 macrophage phagocytosis in a JNK-dependent manner, which could be restored by Box5. In addition, rWnt5a-induced migration of THP-1 macrophages was also reversed by Box5. Our findings suggested that Wnt5a/JNK signaling plays an important role in the development of ALF and that Box5 could have particular hepatoprotective effects in ALF.

Hypermethylation of Cyclin D2 Predicts Poor Prognosis of Hepatitis B Virus-Associated Hepatocellular Carcinoma after Hepatectomy.

Prognosis of patients with hepatocellular carcinoma remains poor because of progression of hepatocellular carcinoma and high recurrence rates. Cyclin D2 (CCND2) plays a vital role in regulating the cell cycle; indeed, aberrant methylation of CCND2 is involved in the development of hepatocellular carcinoma. Therefore, we aimed to investigate levels of CCND2 methylation in patients with hepatitis B virus (HBV)-associated hepatocellular carcinoma and to evaluate its prognostic significance after hepatectomy. In total, 257 subjects were enrolled (166 hepatocellular carcinoma patients undergoing surgical resection, 61 chronic hepatitis B (CHB) patients, and 30 healthy controls). CCND2 methylation in peripheral blood mononuclear cells was measured quantitatively using MethyLight. We found that CCND2 methylation levels in patients with HBV-associated hepatocellular carcinoma were significantly higher than in CHB patients (P < 0.001) or healthy controls (P < 0.001). Within the hepatocellular carcinoma group, CCND2 methylation levels were higher in patients with portal vein invasion, early tumor recurrence, TNM III/IV stage, and tumor size ≥ 5 cm (P < 0.05). Furthermore, higher levels of CCND2 methylation were associated with worse overall survival and disease-free survival (P = 0.005 and P < 0.001, respectively). Multivariate analysis identified CCND2 methylation as an independent prognostic factor for early tumor recurrence (P = 0.021), overall survival (P = 0.022), and disease-free survival (P < 0.001) in hepatocellular carcinoma patients after resection. In conclusion, hypermethylation of CCND2 may have clinical utility for predicting a high risk of poor prognosis and early tumor recurrence in patients with HBV-associated hepatocellular carcinoma after hepatectomy.

Hypermethylation of secreted frizzled related protein 2 gene promoter serves as a noninvasive biomarker for HBV-associated hepatocellular carcinoma.

For patients with hepatocellular carcinoma (HCC), early detection is critical to improve survival. Secreted frizzled-related protein 2 (SFRP2) is a candidate tumor suppressor as Wnt antagonist and SFRP2 promoter has been found hypermethylated in various malignancies. This study aimed to investigate the methylation status of SFRP2 promoter in hepatitis B virus (HBV) associated HCC and estimate its diagnostic value as a non-invasive biomarker. A total of 293 patients, including 132 patients with HBV-associated HCC, 121 with chronic hepatitis B (CHB) and 40 healthy controls (HCs) were enrolled. SFRP2 methylation level in peripheral mononuclear cells (PBMCs) was quantitatively detected by MethyLight. SFRP2 methylation level was significantly higher in patients with HBV-associated HCC than in those with CHB (p < 0.001) and HCs (p < 0.001) while mRNA level of SFRP2 was significantly lower in HCC group than the other two groups (p < 0.05). In HCC subgroup, SFRP2 methylation level markedly increased in patients >50 years old, female, with negative HBeAg, negative HBV-DNA and poor differentiation compared with the remaining groups (P < 0.05). Furthermore, SFRP2 methylation level showed a significantly better diagnostic value than alpha-fetoprotein (AFP) and the combination of AFP and methylation levels of SFRP2 markedly improved the area under the receiver operating characteristic curve (p < 0.05). In conclusion, hypermethylation of SFRP2 promoter exists in HBV-associated HCC. The combination of SFRP2 methylation level in PBMCs and AFP could significantly improve the diagnostic ability of AFP in discriminating HBV-associated HCC from CHB and SFRP2 methylation level had the potential to serve as a non-invasive biomarker for HCC diagnosis.

5-Aza-2'-deoxycytidine may enhance the frequency of T regulatory cells from CD4+ naïve T cells isolated from the peripheral blood of patients with chronic HBV infection.

OBJECTIVES: Methylation pattern of gene modification is essential for the differentiation of T regulatory cells (Tregs) and 5-Aza-2'-deoxycytidine is a common inhibitor of methylation. This study aimed to investigate the potential effects of Treg polarizing conditions and 5-Aza-2'-deoxycytidine treatment in the differentiation of naïve T cells during chronic hepatitis B virus (HBV) infection. METHODS: The frequency of Tregs in peripheral blood was determined by flow cytometry from patients with chronic hepatitis B (CHB) (n = 51), liver cirrhosis (LC) (n = 47), hepatocellular carcinoma (HCC) (n = 40) and healthy controls (HCs) (n = 17). Gene expression were detected by qRT-PCR and DNA methyltransferases (DNMT) Activity was also determined. RESULTS: The frequency of Tregs and Foxp3 expression in peripheral blood from 5-Aza-2'-deoxycytidine-treated groups were higher than that with acetic acid treatment as a control. Foxp3 mRNA and the frequency of Tregs derived from naïve CD4+T cells from peripheral blood of patients with HCC or LC were more pronounced compared with HCs. 5-Aza-2'-deoxycytidine may have induced a more pronounced upward trend of PD-1 expression in HBV patients. CONCLUSIONS: 5-Aza-2'-deoxycytidine mediated demethylation has potential effects on enhancing the differentiation of naïve T cells to Tregs in chronic HBV infection.

Measurement of Cyclin D2 (CCND2) Gene Promoter Methylation in Plasma and Peripheral Blood Mononuclear Cells and Alpha-Fetoprotein Levels in Patients with Hepatitis B Virus-Associated Hepatocellular Carcinoma.

BACKGROUND Alpha-fetoprotein (AFP) is widely used to screen for hepatocellular carcinoma (HCC). However, the use of this biomarker has been challenged due to its low sensitivity and high rate of false negatives. In this study, we evaluated the diagnostic capability of cyclin D2 (CCND2) promoter methylation in patients with HCC related to hepatitis B virus (HBV). MATERIAL AND METHODS Using methylation-specific PCR and quantitative real-time PCR, we measured methylation status and mRNA levels of CCND2 in plasma and peripheral blood mononuclear cells (PBMCs) from 275 subjects: 75 patients with chronic hepatitis B (CHB), 47 with liver cirrhosis (LC), 118 with HCC, and 35 healthy controls (HCs). RESULTS The methylation rate of the CCND2 promoter was significantly higher in HCC patients than in patients without HCC (P<0.001). Furthermore, advanced HCC (TNM III/IV) was associated with a significantly higher frequency of CCND2 methylation and lower CCND2 mRNA levels than early-stage disease (TNM I/II; P<0.05). Combined measurement of CCND2 methylation and AFP yielded significantly higher sensitivity and area under the curve (AUC) than AFP alone in distinguishing patients with HCC from subjects with LC and CHB (P<0.001). CONCLUSIONS CCND2 methylation may be useful for predicting HCC progression. In addition, combined measurement of CCND2 methylation and AFP could serve as a non-invasive diagnostic marker for patients with HBV-related HCC.

Diagnostic Value of the Hypomethylation of the WISP1 Promoter in Patients with Hepatocellular Carcinoma Associated with Hepatitis B Virus.

Wnt1-inducible signaling pathway protein 1 (WISP1) regulates cell proliferation, differentiation, adhesion, migration and survival. Abnormal WISP1 expression is associated with the carcinogenesis of hepatocellular carcinoma (HCC). Aberrant DNA methylation is one of the major epigenetic alterations in HCC. However, the methylation status of the WISP1 promoter is still unclear. We therefore aimed to determine the methylation status of the WISP1 promoter and evaluate its clinical value in HCC. The study enrolled 251 participants, including 123 participants with HCC, 90 participants with chronic hepatitis B (CHB) and 38 healthy controls (HCs). WISP1 methylation status, mRNA levels and plasma soluble WISP1 were detected by methylation-specific polymerase chain reaction (MSP), quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. We found that the methylation frequency of WISP1 in patients with HCC was significantly lower than that in patients with CHB and HCs, while the relative expression levels of WISP1 mRNA were markedly higher in patients with HCC than in patients with CHB and HCs. Furthermore, the plasma soluble WISP1 in patients with HCC was obviously lower than in that in patients with CHB and HCs. Alpha-fetoprotein (AFP) is a widely recognized biomarker to diagnose HCC which lacks enough sensitivity and specificity. WISP1 promoter methylation status combined with AFP significantly improved the diagnostic ability in discriminating HCC from CHB compared with AFP or WISP1 methylation status alone. In conclusion, hypomethylation of the WISP1 gene promoter may serve as a noninvasive biomarker for detecting HBV-associated HCC.

Combined use of murine double minute-2 promoter methylation and serum AFP improves diagnostic efficiency in hepatitis B virus-related hepatocellular carcinoma.

Objective: Hepatocellular carcinoma (HCC) accounts for approximately 85% of all cases of liver cancer. In China, chronic hepatitis B virus-related HCC (HBV-related HCC) is the most common type of HCC. However, the majority of HBV-related HCC patients are asymptomatic, and the best opportunities for treating these patients are missed. The precise diagnosis of HBV-related HCC is crucial. The main purpose of this study was to evaluate the diagnostic value of murine double minute-2 (MDM2) promoter methylation in HBV-related HCC patients. Methods: The methylation status of the MDM2 promoter was detected by methylation-specific PCR. The MDM2 expression levels were validated by quantitative real-time PCR. Enzyme-linked immunosorbent assay was used to determine the levels of interleukin-6 (IL-6) and tumor-necrosis factor-α (TNF-α) in plasma. Results: The methylation frequency of the MDM2 promoter was decreased in HBV-related HCC patients. The MDM2 mRNA levels of patients with HBV-related HCC were higher than those of patients with liver cirrhosis and chronic hepatitis B. The plasma levels of IL-6 and TNF-α were significantly higher in HBV-related HCC patients than that in liver cirrhosis and chronic hepatitis B patients. The TNF-α levels were higher in the unmethylated MDM2 promoter group than in the methylated MDM2 promoter group in HBV-related HCC patients. Moreover, the combination of MDM2 promoter methylation and alpha-fetoprotein (AFP) improved the diagnosis of HBV-related HCC. Conclusions: Our study indicates, for the first time, that MDM2 promoter hypomethylation is present in HBV-related HCC patients. The combination of MDM2 promoter methylation and AFP can greatly improve diagnostic efficiency in HBV-related HCC, which might provide a new method for HBV-related HCC diagnosis.

Radiofrequency ablation versus microwave ablation for early stage hepatocellular carcinoma: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: Several randomized control trials (RCTs) were conducted to compare microwave ablation (MWA) and radiofrequency ablation (RFA) in the treatment of hepatocellular carcinoma (HCC) over the years. The purpose of this study was to compare the efficacy of RFA and MWA for early stage HCC. METHODS: Studies were systematically searched on Emabse, Ovid Medline, PubMed, and Cochrane Library until March 20, 2020. Continuous variables and dichotomous variables were compared using weighted mean difference (WMD) and odds ratio (OR), respectively. For the comparison of overall survival (OS) and disease-free survival (DFS), the hazard ratio (HR) and 95% confidence interval (CI) were extracted from univariate analysis or survival plots. RESULTS: A total of 26 studies (5 RCTs and 21 cohorts) with 4396 patients (2393 patients in RFA and 2003 patients in MWA) were included in our study. Of these patients, 47% received treatment under general anesthesia in the MWA group and 84% in the RFA group (OR = 0.529, P < .001). The median ablation time was reduced in the MWA group (12 minutes) compared with RFA group (29 minutes) (WMD = -15.674, P < .001). In total, 17.6% patients exhibited progression during follow-up in the MWA group compared with 19.5% in the RFA group (OR = 0.877, P = .225). No statistically significant differences were observed between MWA and RFA groups in terms of OS and DFS (HR = 0.891 and 1.014, P = .222 and .852, respectively). CONCLUSIONS: MWA exhibited similar therapeutic effects as RFA in the treatment of early stage HCC. Given the shorter ablation time, MWA can be performed under local anesthesia.

RIPK3 mRNA level acts as a diagnostic biomarker in hepatitis B virus-associated hepatocellular carcinoma.

HBV-associated hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and non-invasive early detection of HBV-associated HCC requires to be improved. To determine the alteration and clinical relevance of necroptosis and its key regulator receptor-interacting protein kinase 3 (RIPK3) in HBV-associated HCC, we detected the mRNA level of RIPK3 in peripheral blood mononuclear cells (PBMCs) and analyzed its correlation with clinical parameters. Here, we demonstrate that the expression of RIPK3 is elevated in patients with HBV-associated HCC compared to patients with chronic hepatitis B (CHB) and patients with HBV-related liver cirrhosis (LC). The mRNA level of RIPK3 is positively correlated with the severity of clinical manifestations and TNM stages. Moreover, the serum levels of RIPK3-asssocited cytokines are altered in consistent with the change of RIPK3 expression. The diagnostic accuracy of RIPK3 mRNA level is comparable to AFP test in discriminating HBV-associated HCC from LC and is better than AFP test in discriminating HBV-associated HCC from CHB. The combination of RIPK3 mRNA level and AFP test significantly improves the diagnosis of HBV-associated HCC. These data suggest that RIPK3 mRNA level is a biomarker in the onset and progression of HBV-associated HCC and may provide novel diagnostic strategies combined with the AFP test.

Hypomethylation of the cyclin D1 promoter in hepatitis B virus-associated hepatocellular carcinoma.

The hypomethylation of the Cyclin D1 (CCND1) promoter induced by excess oxidative stress likely promotes the development of hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC). We aimed to evaluate methylation status of the CCND1 promoter as a new plasma marker for the detection of HBV-HCC.We consecutively recruited 191 participants, including 105 patients with HBV-HCC, 54 patients with chronic hepatitis B (CHB), and 32 healthy controls (HCs). Using methylation-specific polymerase chain reaction, we identified the methylation status of the CCND1 promoter in plasma samples. We analyzed the expression levels of the CCND1 mRNA in peripheral blood mononuclear cells by using quantitative real-time PCR. We assessed the plasma levels of superoxide dismutase, 8-hydroxydeoxyguanosine and malondialdehyde by using enzyme-linked immunosorbent assays.Patients with HBV-HCC (23.81%) presented a reduced methylation frequency compared with patients with CHB (64.81%) or HCs (78.13%) (P < .001). When receiver operating characteristic curves were plotted for patients with HBV-HCC versus CHB, the methylation status of the CCND1 promoter yielded diagnostic parameter values for the area under the curve of 0.705, sensitivity of 76.19%, and specificity of 64.81%, thus outperforming serum alpha-fetoprotein (AFP), which had an area under the curve of 0.531, sensitivity of 36.19%, and specificity of 90.74%. Methylation of the CCND1 promoter represents a prospective diagnostic marker for patients with AFP-negative HBV-HCC and AFP-positive CHB. The expression levels of CCND1 mRNA was increased in patients with HBV-HCC compared with patients with CHB (Z = -4.946, P < .001) and HCs (Z = -6.819, P < .001). Both the extent of oxidative injury and antioxidant capacity indicated by the superoxide dismutase, 8-hydroxydeoxyguanosine and malondialdehyde levels were increased in patients with HBV-HCC. Clinical follow up of patients with HBV-HCC revealed a worse overall survival (P = .012, log-rank test) and a decreased progression-free survival (HR = 0.109, 95%CI: 0.031-0.384) for the unmethylated CCND1 group than methylated CCND1 group.Our study confirms that oxidative stress appears to correlate with plasma levels of CCND1 promoter methylation, and the methylation status of the CCND1 promoter represents a prospective biomarker with better diagnostic performance than serum AFP levels.

Methylation status of the T-cadherin gene promotor in peripheral blood mononuclear cells is associated with HBV-related hepatocellular carcinoma progression.

DNA methylation is one of the epigenetic mechanisms to regulate gene expression and frequently occurs in human cancer cells. T-cadherin (CDH13) is a new member of the cadherin superfamily and possesses multiple functions. Our study included 26 normal controls (NCs), 65 chronic hepatitis B patients (CHB), 14 liver cirrhosis patients (LC) and 157 hepatocellular carcinoma patients (HCC). We mainly focused on the mRNA expression and methylation status of CDH13 in peripheral blood mononuclear cells (PBMCs), which were detected by semi-quantitative real-time polymerase chain reaction (RT-qPCR) and methylation-specific polymerase chain reaction (MSP) respectively. The CDH13 mRNA level was lower in HCC, especially in early-stage of HCC than in NCs and CHB groups (p < 0.05). Methylation frequency of the CDH13 promoter was significantly higher in HCC patients than in the NCs and CHB groups (67.52 % vs 0.00 %, p < 0.001, 67.52 % vs 52.31 %, p < 0.05, respectively). CDH13 mRNA level was significantly and relatively lower in methylated groups than in unmethylated groups among the whole participants. The methylation level of CDH13 promoter in HCC might be influenced or partly influenced by some critical factors such as TBil, ALB and AFP (p < 0.05). As an important factor in signaling pathway regulating by CDH13 to promote carcinogenesis, JNK level was significantly higher in HCC which had a higher methylation frequency than in NCs, CHB and LC (p < 0.05). Furthermore, the combination of the methylated CDH13 level and AFP level showed a better score: AUC = 0.796 (SE = 0.031, 95 %CI 0.735-0.857; p < 0.001) in male and AUC = 0.832 (SE = 0.057, 95 %CI 0.721-0.944; p < 0.001) in female compared to AFP alone for diagnosing HCC from NCs, CHB and LC. The methylation of CDH13 promoter was an independent predictor for assessing the prognosis of HCC patients (r=-1.378 p < 0.05). In conclusion, hypermethylation of CDH13 in PBMCs was associated with the underexpression of mRNA and the high risk of HCC. The methylation status of the CDH13 promoter in PBMCs was a potential noninvasive biomarker to predict the prognosis of HCC patients.

TNFRSF12A and a new prognostic model identified from methylation combined with expression profiles to predict overall survival in hepatocellular carcinoma.

BACKGROUND: It has been proved that DNA methylation, as an epigenetic regulatory mode, plays a crucial role in the initiation, progression and invasion of hepatocellular carcinoma (HCC). However, there still are some pathways and factors that regulates the carcinogenesis of HCC remains unclear. METHODS: The original datasets comparing DNA methylation, clinical information and transcriptome profiling between HCC and normal controls were downloaded from The Cancer Genome Atlas (TCGA) database. R software was used to screen for methylation-differential genes (MDGs) and methylation-driven genes. Gene-functional enrichment analysis, ConsensusPathDB pathway analysis, protein-protein interaction (PPI) network construction and survival analysis were performed; methylation-specific polymerase chain reaction (MSP) and real-time quantitative polymerase chain reaction (RT-qPCR) were used for validation. RESULTS: One hundred and sixty-seven MDGs and 285 methylation-driven genes were identified. Function and pathway enrichment analysis revealed that they are associated with sequence-specific DNA binding, nuclear nucleosome, regulation of insulin-like growth factor transport, etc. An eight-gene (HIST1H1D, RP11-476B1.1, OR2AK2, TNFRSF12A, CTD-2313N18.8, AC133644.2, RP11-467L13.4 and LINC00989) prognostic model was identified from the MDGs; its methylation degree can strongly predict the overall survival of HCC. Among them, TNFRSF12A being the only one belongs to both MDGs and methylation-driver genes, shows a significant independent correlation with the prognosis of HCC. That was validated in further details. CONCLUSIONS: Our research has identified a registry of novel genes and pathways that's important for regulating the carcinogenesis of HCC. In addition, we identified a strong molecular model for prognostic prediction. These findings will not only provide guidance for clinical individualized treatment, but also to set us targets for further research on the molecular mechanism of HCC.

Tumor necrosis factor-α-induced protein 8-like 2 mRNA in peripheral blood mononuclear cells is associated with the disease progression of chronic hepatitis B virus infection.

BACKGROUND: Tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) is a novel target and molecule in the negative regulation of immune homeostasis. The present study aimed to investigate the dynamic expression of TIPE2 mRNA during the progression of chronic hepatitis B virus (HBV) infection. METHODS: A total of 193 patients with chronic HBV infection were retrospectively recruited into this cross-sectional study, including 97 patients with chronic hepatitis B (CHB), 55 with liver cirrhosis and 41 with HBV-related hepatocellular carcinoma (HCC). TIPE2 mRNA was determined using real-time quantitative polymerase chain reaction. RESULTS: The expression of TIPE2 levels in patients with HCC was significantly decreased compared with expression in patients with liver cirrhosis, CHB and healthy controls (P < 0.05); meanwhile, the TIPE2 mRNA levels in patients with CHB and liver cirrhosis were significantly increased compared with levels in healthy controls (P < 0.01). In liver cirrhosis, the TIPE2 mRNA level in the decompensated state was significantly higher than that in the compensated state (P < 0.05). In HCC patients, TIPE2 mRNA was significantly associated with venous invasion, tumor size and tumor node metastasis stage. Furthermore, the optimal cutoff of 0.78 for the level of TIPE2 mRNA has a sensitivity of 97.56% and a specificity of 88.16% for discriminating HCC from patients with CHB and liver cirrhosis. CONCLUSIONS: TIPE2 mRNA was associated with various stages of chronic HBV infection, ranging from CHB to liver cirrhosis and HCC. Furthermore, TIPE2 mRNA with an optional cutoff value of 0.78 might serve as a promising biomarker to discriminate HBV-associated HCC from CHB and LC patients.