Systematic Analysis Identifies a Specific RNA-Binding Protein-Related Gene Model for Prognostication and Risk-Adjustment in HBV-Related Hepatocellular Carcinoma.

OBJECTIVE: Increasing evidence shows that dysregulated RNA binding proteins (RBPs) modulate the progression of several malignancies. Nevertheless, their clinical implications of RBPs in HBV-related hepatocellular carcinoma (HCC) remain largely undefined. Here, this study systematically analyzed the associations of RBPs with HBV-related HCC prognosis. METHODS: Based on differentially expressed RBPs between HBV-related HCC and control specimens, prognosis-related RBPs were screened by univariate analyses. A LASSO model was then created. Kaplan-Meier curves, ROCs, multivariate analyses, subgroup analyses and external verification were separately applied to assess the efficacy of this model in predicting prognosis and recurrence of patients. A nomogram was created by incorporating the model and clinical indicators, which was verified by ROCs, calibration curves and decision curve analyses. By CIBERSORT algorithm, the association between the risk score and immune cell infiltrations was evaluated. RESULTS: Totally, 54 RBPs were distinctly correlated to prognosis of HBV-related HCC. An 11-RBP model was created, containing POLR2L, MRPS12, DYNLL1, ZFP36, PPIH, RARS, SRP14, DDX41, EIF2B4, and NOL12. This risk score sensitively and accurately predicted one-, three- and five-year overall survival, disease-free survival, and progression-free interval. Compared to other clinical parameters, this risk score had the best predictive efficacy. Also, the clinical generalizability of the model was externally verified in the GSE14520 dataset. The nomogram may predict patients' survival probabilities. Also, the risk score was related to the components in the immune microenvironment. CONCLUSION: Collectively, RBPs may act as critical elements in the malignant progression of HBV-related HCC and possess potential implications on prognostication and therapy decision.

Clinical implications of serum hepatitis B virus RNA quantitation in untreated chronic hepatitis B virus-infected patients.

Serum hepatitis B virus (HBV) RNA quantitation may be useful for managing untreated chronic HBV-infected patients, but its distribution characteristics and relationship to HBV DNA are unclear. A retrospective cohort including 149 untreated HBV-infected patients was divided into four clinical phenotypes: hepatitis B envelope antigen (HBeAg) positive with normal alanine transaminase (ALT; EPNA) or with elevated ALT (EPEA), HBeAg-negative with normal ALT (ENNA) or with elevated ALT (ENEA). Serum HBV RNA levels were quantified by a high-sensitivity real-time fluorescent quantitative PCR method and liver biopsy was performed in those with undetectable serum HBV DNA or RNA. The detectable serum HBV RNA levels (log10 copies/mL) in EPNA, EPEA, ENNA, and ENEA were 6.02±1.48, 6.54±1.27, 2.51±0.78 and 3.54±1.25, respectively. The low level (< 2.0 log10 copies/mL) comprised mainly of ENNA phenotype (76.9%), while the high level (> 6.0 log10 copies/mL) was HBeAg-positive patients (98.1%). Serum HBV RNA level were significantly correlated with serum HBV DNA and HBsAg in HBeAg-positive phenotypes, but a correlation only with HBV DNA was observed in ENEA patients. Serum HBV DNA and RNA were both independent risk factors associated with elevated ALT in HBeAg-negative patients. Seven serum HBV DNA-undetectable but RNA-detectable patients underwent liver biopsy, showing moderate or severe liver inflammation. Varying serum HBV RNA levels can reflect natural disease phases in untreated HBV-infected patients, indicating that this biomarker could reflect liver inflammation in untreated HBeAg-negative patients as successfully as serum HBV DNA. Serum HBV RNA can complement clinical management strategies when serum HBV DNA is undetectable.

Corosolic Acid Inhibits Cancer Progress Through Inactivating YAP in Hepatocellular Carcinoma.

Chemotherapy is critical for the treatment of hepatocellular carcinoma (HCC). Despite the proapoptotic effects of corosolic acid (CA) treatment, its underlying mechanism is not completely clear. The aim of this study was to determine the molecular mechanism of CA in HCC treatment. MTT assay was used to determine the IC50 of CA. Immunoprecipitation and immunofluorescence were used to detect the interaction and subcellular localization of Yes-associated protein (YAP) and mouse double minute 2 (MDM2). In addition, in vivo xenotransplantation was performed to assess the effects of CA, YAP, and MDM2 on tumorigenesis. The IC50 of CA was about 40 M in different HCC cell lines, and CA decreased YAP expression by reducing its stability and increasing its ubiquitination. CA treatment and MDM2 overexpression significantly decreased the crosstalk between YAP and cAMP-responsive element-binding protein (CREB), TEA domain transcription factor (TEAD), and Runt-related transcription factor 2 (Runx2). CA stimulation promoted the translocation of YAP and MDM2 from the nucleus to the cytoplasm and increased their binding. In addition, CA treatment obviously reduced tumorigenesis, whereas this effect was abolished when cells were transfected with sh-MDM2 or Vector-YAP. The present study uncovered that CA induced cancer progress repression through translocating YAP from the nucleus in HCC, which might provide a new therapeutic target for HCC.

Upexpression of BHLHE40 in gastric epithelial cells increases CXCL12 production through interaction with p-STAT3 in Helicobacter pylori-associated gastritis.

BHLHE40, a member of the basic helix-loop-helix transcription factor family, has been reported to play an important role in inflammatory diseases. However, the regulation and function of BHLHE40 in Helicobacter pylori (H pylori)-associated gastritis is unknown. We observed that gastric BHLHE40 was significantly elevated in patients and mice with H pylori infection. Then, we demonstrate that H pylori-infected GECs express BHLHE40 via cagA-ERK pathway. BHLHE40 translocates to cell nucleus, and then binds to cagA protein-activated p-STAT3 (Tyr705). The complex increases chemotactic factor CXCL12 expression (production). Release of CXCL12 from GECs fosters CD4+ T cell infiltration in the gastric mucosa. Our results identify the cagA-BHLHE40-CXCL12 axis that contributes to inflammatory response in gastric mucosa during H pylori infection.

Pretreatment microRNA levels can predict HBsAg clearance in CHB patients treated with pegylated interferon α-2a.

BACKGROUND: To investigate the predictive capability of microRNAs (miRNAs) prior treatment for HBsAg clearance in chronic hepatitis B (CHB) treated with pegylated interferon α-2a (PEG-IFNα-2a). METHODS: The treatment effect was determined by HBsAg clearance and subjects were classified into HBsAg clearance group and non HBsAg clearance group. Differential miRNAs expression in peripheral blood mononuclear cells (PBMC) was screened using microarrays in an identification cohort (n = 20) and validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in a confirmation cohort (n = 47). Receiver operating characteristic curve (ROC), logistic regression and gene ontology (GO)/Pathway analyses were used to evaluate the predictive capability of selected miRNAs for HBsAg clearance and determine their mechanistic roles. RESULTS: Twenty-seven subjects (40.3%) acquired HBsAg clearance, ten in the identification cohort and seventeen in the confirmation cohort. Four miRNAs out of twelve (miR-3960, miR-126-3p, miR-335-5p, miR-23a-3p) were verified to be differential expressed by qRT-PCR in the confirmation cohort. Their expression patterns were consistent with the microarray results. Their levels were lower in the response group compared with the nonresponse group (p < 0.05). The areas under curve (AUC) were 0.8333 (p = 0.001), 0.751 (p = 0.01), 0.7294 (p = 0.013), 0.6275 (p = 0.094) and positive predict values (PPV) were 84.62, 60.00, 70.00, 28.57% for miR-3960, miR-126-3p, miR-335-5p, and miR-23a-3p respectively. The AUC and PPV of the combination of miR-3960 and miR-126-3p were 0.8529 and 92.31%, which were better than using miR-3960 alone, but the differences were not statistically significance (p > 0.05). CONCLUSIONS: We identified differential expressed miRNAs between response and nonresponse groups of PEG-IFNα-2a treatment and demonstrated that miR-3960 was the optimal predictor for HBsAg clearance compared with other miRNAs, but it requires to be further comfired in larger cohort studies. TRIAL REGISTRATION: ChiCTR ChiCTR-ROC-16008735, registered retrospectively on 28 June, 2016.

Performance of several simple, noninvasive models for assessing significant liver fibrosis in patients with chronic hepatitis B.

AIM: To compare the performance of several simple, noninvasive models comprising various serum markers in diagnosing significant liver fibrosis in the same sample of patients with chronic hepatitis B (CHB) with the same judgment standard. METHODS: A total of 308 patients with CHB who had undergone liver biopsy, laboratory tests, and liver stiffness measurement (LSM) at the Southwest Hospital, Chongqing, China between March 2010 and April 2014 were retrospectively studied. Receiver operating characteristic (ROC) curves and area under ROC curves (AUROCs) were used to analyze the results of the models, which incorporated age-platelet (PLT) index (API model), aspartate transaminase (AST) to alanine aminotransferase (ALT) ratio (AAR model), AST to PLT ratio index (APRI model), γ-glutamyl transpeptidase (GGT) to PLT ratio index (GPRI model), GGT-PLT-albumin index (S index model), age-AST-PLT-ALT index (FIB-4 model), and age-AST-PLT-ALT-international normalized ratio index (Fibro-Q model). RESULTS: The AUROCs of the S index, GPRI, FIB-4, APRI, API, Fibro-Q, AAR, and LSM for predicting significant liver fibrosis were 0.726 (P<0.001), 0.726 (P<0.001), 0.621 (P=0.001), 0.619 (P=0.001), 0.580 (P=0.033), 0.569 (P=0.066), 0.495 (P=0.886), and 0.757 (P<0.001), respectively. The S index and GPRI had the highest correlation with histopathological scores (r=0.373, P<0.001; r=0.372, P<0.001, respectively) and LSM values (r=0.516, P<0.001; r=0.513, P<0.001, respectively). When LSM was combined with S index and GPRI, the AUROCs were 0.753 (P<0.001) and 0.746 (P<0.001), respectively. CONCLUSION: S index and GPRI had the best diagnostic performance for significant liver fibrosis and were robust predictors of significant liver fibrosis in patients with CHB for whom transient elastography was unavailable.

Interaction of TLR-IFN and HLA polymorphisms on susceptibility of chronic HBV infection in Southwest Han Chinese.

BACKGROUND & AIMS: The toll-like receptor-interferon (TLR-IFN) signalling pathway plays a crucial role in HBV infection. Human leucocyte antigen (HLA) polymorphisms are associated with chronic HBV infection by genome wide association study (GWAS). We aimed to explore interaction between TLR-IFN and HLA gene polymorphisms in susceptibility of chronic HBV infection. METHODS: In the Chinese Southwest Han population, 1191 chronic HBV infection patients and 273 HBV clearance were selected. A total of 39 single nucleotide polymorphism loci in 23 genes of the TLR-IFN pathway and four HLA polymorphism loci associated with chronic HBV infection identified by GWAS were selected for genotyping. SNPStats, QVALUE, and multifactor dimensionality reduction were used for statistical analysis. RESULTS: A significant association was seen in several of the TLR-IFN pathway genes, TLR9 rs352140 (OR = 0.70, P = 0.0088), IL1B rs16944 (OR = 0.67, P = 0.016), IL12B rs3212227 (OR = 1.38, P = 0.021), IFNGR1 rs3799488 (OR = 1.48, P = 0.0048), IFNGR2 rs1059293 (OR = 0.27, P = 0.011), MX1 rs467960 (OR = 0.68, P = 0.022), as well as four loci in HLA, rs3077 (OR = 0.55, P < 0.0001), rs2856718 (OR = 0.60, P = 4e-04), rs9277535 (OR = 0.54, P < 0.0001) and rs7453920 (OR = 0.43, P < 0.0001). A synergistic relationship was seen between rs9277535 and rs16944 (0.13%), rs1143623 and rs6613 (0.10%). The combination of rs9277535 in HLA and rs16944 in IL1B was the best model to predict chronic HBV infection (testing accuracy = 0.6040, P = 0.0010, cross-validation consistency = 10/10). CONCLUSIONS: TLR-IFN pathway gene polymorphisms are associated with chronic HBV infection. Interactions with polymorphisms in these genes may be one mechanism by which HLA polymorphisms influence susceptibility to chronic HBV infection, as specific single nucleotide polymorphism combinations are highly predictive of chronic HBV infection.

Clinical implications of hepatitis B surface antigen quantitation in the natural history of chronic hepatitis B virus infection.

BACKGROUND: HBsAg quantitation may be useful for managing patients with hepatitis B virus (HBV) infection. OBJECTIVES: We explored the clinical implications of HBsAg quantitation for patients with HBsAg levels >250IU/ml (Abbott Diagnostics). STUDY DESIGN: Two hundred and thirty-three HBV-infected patients comprising 29 immune tolerance cases, 49 treatment-naïve HBeAg-positive chronic hepatitis B (CHB) cases, 91 inactive HBV carrier cases, and 64 treatment-naïve HBeAg-negative CHB cases were analyzed. HBsAg was quantified by the Architect HBsAg assay (Abbott Diagnostics) after a 1:500 automated dilution. RESULTS AND CONCLUSIONS: HBsAg (log10IU/ml) was established for immune tolerance (4.50±0.43), HBeAg-positive CHB (4.17±0.66), inactive HBV carrier (3.32±0.44), and HBeAg-negative CHB (3.23±0.40); (p=4.92×10(-35)). No significant difference was observed between inactive HBV carrier and HBeAg-negative CHB (p=0.247). The proportions of HBsAg <2000IU/ml for inactive HBV carrier and HBeAg-negative CHB were 51.6% and 59.3%, respectively (p=0.341). Positive correlations between HBsAg and HBV DNA were observed for immune tolerance (p=1.23×10(-4)) and HBeAg-positive CHB (p=0.003), but not for HBeAg-negative CHB (p=0.432). A negative correlation between HBsAg and age was observed for immune tolerance (p=0.030), HBeAg-positive CHB (p=0.016), and inactive HBV carrier (p=0.001), but not in HBeAg-negative CHB (p=0.249). No significant differences between HBsAg and ALT for HBeAg-positive (p=0.338) or HBeAg-negative CHB (p=0.564) were observed. For patients with HBsAg quantitation >250IU/ml, HBsAg may reflect HBV DNA replication for HBeAg-positive cases. HBsAg is not a suitable marker for evaluating hepatitis activity and distinguishing between cases of HBeAg-negative CHB and inactive HBV carrier state.

Long-term outcomes after nucleos(t)ide analogues discontinuation in chronic hepatitis B patients with HBeAg-negative.

BACKGROUND: Hepatitis B e Antigen (HBeAg)-negative chronic hepatitis B (CHB) patients have an active liver disease with a high risk of progression to decompensated cirrhosis and hepatocellular carcinoma. The management strategy for HBeAg-negative CHB patients treated with nucleos(t)ide analogues (NUCs) is a topic of concern. To observe the outcomes for this population after NUCs withdrawal, HBeAg-negative CHB patients with loss of hepatitis B surface antigen (HBsAg) or sustained undetectable HBV DNA levels who had discontinued NUCs therapy were included in the study. METHODS: A total of 66 patients (2 patients with HBsAg loss and 64 patients with sustained undetectable HBV DNA levels) were examined. HBV DNA levels and alanine aminotransferase (ALT) levels were monitored regularly after discontinuation of NUCs therapy. Relapse was defined as HBV DNA levels >2,000 IU/mL while off therapy in at least two determinations more than 4 weeks apart. RESULTS: The time to achieve undetectable HBV DNA levels was 14 weeks (interquartile range (IQR): 12-24 weeks). The time until consolidation therapy was 144 weeks (IQR: 96-168 weeks). No relapses occurred in either of the HBsAg loss patients. Among the 64 patients with undetectable HBV DNA levels, 19 (29.7%) patients demonstrated evidence of relapse. All the relapses occurred within 96 weeks after discontinuation. The median duration of relapse was 36 weeks (IQR: 12-48 weeks). Elevation of HBV DNA and ALT levels over baseline was only observed in 10% of the relapse patients. There were no significant differences among the baseline characteristics (sex, HBV genotype, age, or ALT level) or the time until consolidation therapy between relapse and sustained-response patients. CONCLUSIONS: NUC discontinuation is feasible after achieving undetectable HBV DNA levels in HBeAg-negative CHB patients. Prolonging the time until consolidation therapy may be a good strategy to decrease the rate of relapse. More than 96 weeks of sustained response is a predictive marker of long-term sustained response.

Expression pattern of serum cytokines in hepatitis B virus infected patients with persistently normal alanine aminotransferase levels.

PURPOSE: About 60-80 % of chronic hepatitis B virus (HBV) carriers are characterized with persistently normal alanine transaminase (ALT). Differences of cytokine expression are associated with the prognosis of HBV infection. We investigated the expression pattern of 30 cytokines associated with anti-HBV immunity in patients with normal ALT. METHODS: Four patient groups (immune tolerance, inactive hepatitis B surface antigen carriers, resolved hepatitis B, and control; 10 subjects per group) were assigned. Thirty cytokines, including IFN-γ, IL-1ß, IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-15, IL-17A, IL-17C, IL-21, IL-22, IL-23p19, IL-28A, IL-29, CCL5, CCL16, CCL20, CCL22, CXCL9, CXCL10, CXCL11, TNFRSF8, TNFRSF18, IL-6R, gp130, and TGF-ß1, were measured using a human cytokine antibody array. Signal intensities were obtained by laser scanner. Protein-protein interactions were analyzed by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). RESULTS: Significant differences of signal intensities were observed for IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-15, IL-21, IL-23p19, IL-28A, and IL-29. The lowest intensity was in controls. Among three HBV infection groups, significant differences were observed in IL-2, IL-4, IL-12p70, IL-15, IL-21, IL-23p19, and IL-29. The highest intensity was in the inactive group. All cytokines with significant differences were involved JAK-STAT signaling that up-regulate FOXP3, SOCS3 and MX1. CONCLUSION: Differential expression of cytokines in JAK-STAT signaling is an important factor associated with prognosis of HBV infection. The elevation of γC cytokines, IL-12p70, IL-23p19, and IL-29 may promote spontaneous HBeAg seroconversion and HBV clearance.