Integrating single-cell and bulk sequencing data to identify glycosylation-based genes in non-alcoholic fatty liver disease-associated hepatocellular carcinoma.

Background: The incidence of non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) has been increasing. However, the role of glycosylation, an important modification that alters cellular differentiation and immune regulation, in the progression of NAFLD to HCC is rare. Methods: We used the NAFLD-HCC single-cell dataset to identify variation in the expression of glycosylation patterns between different cells and used the HCC bulk dataset to establish a link between these variations and the prognosis of HCC patients. Then, machine learning algorithms were used to identify those glycosylation-related signatures with prognostic significance and to construct a model for predicting the prognosis of HCC patients. Moreover, it was validated in high-fat diet-induced mice and clinical cohorts. Results: The NAFLD-HCC Glycogene Risk Model (NHGRM) signature included the following genes: SPP1, SOCS2, SAPCD2, S100A9, RAMP3, and CSAD. The higher NHGRM scores were associated with a poorer prognosis, stronger immune-related features, immune cell infiltration and immunity scores. Animal experiments, external and clinical cohorts confirmed the expression of these genes. Conclusion: The genetic signature we identified may serve as a potential indicator of survival in patients with NAFLD-HCC and provide new perspectives for elucidating the role of glycosylation-related signatures in this pathologic process.

The genetic polymorphism down-regulating HLA-DRB1 enhancer activity facilitates HBV persistence, evolution and hepatocarcinogenesis in the Chinese Han population.

Genetic predisposition of human leucocyte antigen (HLA)-DR has been linked to nonresponse to hepatitis B virus (HBV) vaccination. We sought to reveal their effects on chronic infection and evolution of HBV and development of hepatocellular carcinoma (HCC). Genetic polymorphisms at HLA-DR enhancer regions were genotyped in 4588 participants using quantitative PCR. HBV mutations were determined by sequencing. A dual-luciferase assay was applied to detect the enhancer activity. Associations between HLA-DR polymorphisms and postoperative prognosis were investigated in another cohort of 397 HBV-infected HCC patients. Variant alleles (rs3135395-T, rs3135338-C and rs477515-T) were significantly associated with a decreased risk of HBV persistence in Chinese patients. rs3135395-T, rs3135338-C, rs477515-T and rs2395178-G also significantly decreased HCC risk. rs3135395-T, rs477515-T and rs2395178-G were inversely associated with the generation of A1762T/G1764A, T1753V and C1653T, the HCC-risk HBV mutations. Multiplicative interactions of the variant genotypes with the HCC-risk HBV mutations were significantly associated with a decreased risk of HCC. In multivariate Cox analysis, rs477515-T independently predicted a favourable prognosis, with a hazard ratio of 0.48 (P = .002). The activity of the HLA-DRB1 enhancer with rs477515-T was significantly higher than that with rs477515-C. The activity of the HLA-DRB1 enhancer with rs477515-T and that with rs477515-C was significantly up-regulated by interferon-γ and interleukin-4, respectively. Interleukin-6 significantly inhibited the HLA-DRB1 enhancer activity, and this effect was more evident in those carrying rs477515-T. Polymorphisms predisposing to down-regulation of HLA-DR facilitate the Th1-to-Th2 transition and promote HCC development, possibly via selecting the HCC-risk HBV mutations. This can be transformed into specific prophylaxis of HCC.

Hepatocellular carcinoma with worsened hypoglycemia after transarterial chemoembolization: a case report and systematic review.

Non-islet cell tumor hypoglycemia (NICTH) is an extremely uncommon and serious complication of hepatocellular carcinoma (HCC). Here, we reported a case of a 47-year-old male patient with moderate to poorly differentiated HCC complicated by hypoglycemia that worsened after transarterial chemoembolization (TACE). The patient was admitted into The First Affiliated Hospital of Naval Medical University due to fatigue, nausea, dizziness and passage of tea colored urine. He was diagnosed with NICTH induced by HCC according to CT scanning and laboratory tests. TACE was used as the primary therapy but the hypoglycemia worsened afterward. Then the patient received a liver transplantation as a possible radical cure and hypoglycemia was resolved. We systematically review the management of hypoglycemia caused by HCC and the results show that patients undergoing treatment that mainly alleviate tumor burdens obtained a significantly higher response rate than patients undergoing therapies mainly regulating biologic functions (50.0% vs 27.3%). Cytoreductive surgery, TACE and radiotherapy which aimed to alleviate tumor burdens are effective therapies have great potential, but the risk of hypoglycemic deterioration requires particular attention when using these treatments, especially with TACE.

Genetic Polymorphisms Predisposing the Interleukin 6-Induced APOBEC3B-UNG Imbalance Increase HCC Risk via Promoting the Generation of APOBEC-Signature HBV Mutations.

PURPOSE: APOBEC3-UNG imbalance contributes to hepatitis B virus (HBV) inhibition and somatic mutations. We aimed to explore the associations between hepatocellular carcinoma (HCC) risk and genetic polymorphisms predisposing the imbalance.Experimental Design: Genetic polymorphisms at APOBEC3 promoter and UNG enhancer regions were genotyped in 5,621 participants using quantitative PCR. HBV mutations (nt.1600-nt.1945, nt.2848-nt.155) were determined by Sanger sequencing. Dual-luciferase reporter assay was applied to detect the transcriptional activity. Effects of APOBEC3B/UNG SNPs and expression levels on HCC prognosis were evaluated with a cohort of 400 patients with HCC and public databases, respectively. RESULTS: APOBEC3B rs2267401-G allele and UNG rs3890995-C allele significantly increased HCC risk. rs2267401-G allele was significantly associated with the generation of APOBEC-signature HBV mutation whose frequency consecutively increased from asymptomatic HBV carriers to patients with HCC. Multiplicative interaction of rs2267401-G allele with rs3890995-C allele increased HCC risk, with an adjusted OR (95% confidence interval) of 1.90 (1.34-2.81). rs2267401 T-to-G and rs3890995 T-to-C conferred increased activities of APOBEC3B promoter and UNG enhancer, respectively. IL6 significantly increased APOBEC3B promoter activity and inhibited UNG enhancer activity, and these effects were more evident in those carrying rs2267401-G and rs3890995-C, respectively. APOBEC3B rs2267401-GG genotype, higher APOBEC3B expression, and higher APOBEC3B/UNG expression ratio in HCCs indicated poor prognosis. APOBEC-signature somatic mutation predicts poor prognosis in HBV-free HCCs rather than in HBV-positive ones. CONCLUSIONS: Polymorphic genotypes predisposing the APOBEC3B-UNG imbalance in IL6-presenting microenvironment promote HCC development, possibly via promoting the generation of high-risk HBV mutations. This can be transformed into specific prophylaxis of HBV-caused HCC.

Utility and Safety of Tolvaptan in Cirrhotic Patients with Hyponatremia: a Prospective Cohort Study

Abstract: Introduction and aim. Hyponatremia is common in patients with decompensated cirrhosis and is associated with increased mortality. Tolvaptan, a vasopressor V2 receptor antagonist, can increase free wáter excretion, but its efficacy and safety in cirrhotic patients remain unclear. Material and methods. We studied the usage and safety of tolvaptan in cirrhotic patients in a real-life, non-randomized, multicenter prospective cohort study. Forty-nine cirrhotic patients with hyponatremia were treated with tolvaptan 15 mg daily, and 48 patients not treated with tolvaptan in the same period served as controls. Improvement in serum sodium level was defined as an increase in serum sodium from < 125 to ≥ 125 mmol/L or from 125-134 to ≥ 135 mmol/L on day 7. Results. Twenty-three (47%) patients in the tolvaptan group and 17 (35%) in the control group had normal serum sodium on day 7 (p = 0.25). Serum sodium improved in 30 (61%) patients in the tolvaptan group and 17 (35%) patients in the control group (p = 0.011). Adverse events occurred in 46-47% of patients in both groups, and tolvaptan was not associated with worsened liver function. No patient with normal serum sodium on day 7 died within 30 days of treatment, whereas 16% of those with persistent hyponatremia died (p = 0.0019). Conclusion. In conclusion, short-term tolvaptan treatment is safe and can improve serum sodium level in cirrhotic patients with hyponatremia. Normalization of serum sodium level is associated with better survival.

HCV core protein-induced upregulation of microRNA-196a promotes aberrant proliferation in hepatocellular carcinoma by targeting FOXO1.

The hepatitis C virus (HCV) core protein is critical in the development of hepatocellular carcinoma (HCC). Investigations on HCC have previously focused on microRNAs, a class of small non­coding RNAs, which are crucial in cancer development and progression. The present study aimed to investigate whether microRNA (miR)­196a is aberrantly regulated by the HCV core protein, and whether miR­196a is involved in the regulation of the aberrant proliferation of HCV­HCC cells. In the study, miRNA expression was detected by quantitative polymerase chain reaction analysis. An Ad­HCV core adenovirus was constructed and cell proliferation was measured using a Cell Counting Kit-8 assay and a cell cycle assay following infection. The results of the present study demonstrated that the HCV core protein increased the expression of miR­196a, and that overexpression of miR­196a in the HepG2 and Huh­7 HCC cell lines promoted cell proliferation by inducing the G1­S transition. Furthermore, the present study demonstrated that forkhead box O1 (FOXO1) was directly regulated by miR­196a, and was essential in mediating the biological effects of miR­196a in HCC. The overexpression of FOXO1 markedly reversed the effect of miR­196a in HCC cell proliferation. Taken together, the data obtained in the present study provided compelling evidence that elevated expression levels of miR­196a by the HCV core protein can function as an onco­microRNA during HCV­induced cell proliferation by downregulating the expression of FOXO1, indicating a potential novel therapeutic target for HCV-related HCC.

[Cholestasis morbidity rate in first-hospitalized patients with chronic liver disease in Shanghai].

OBJECTIVE: To investigate the epidemiological status of cholestasis in first-hospitalized patients with chronic liver disease in Shanghai, and to provide a scientific basis for developing prevention and treatment measures. METHODS: From April 2005 to September 2014, 5,146 first-hospitalized patients in Shanghai with a diagnosis of chronic liver disease were enrolled in this study. Clinical data of the 4,660 patients who fit the study criteria for participation were collected for retrospective analysis.Diagnosis of cholestasis was made according to serum alkaline phosphatase (ALP) levels higher than 1.5 times the upper limit normal (ULN) and gamma-glutamyltransferase (GGT) levels higher than 3 times the ULN. The incidence rate of cholestasis was assessed for relation to age, sex, etiology, and type of liver disease, and statistically compared to the general clinical data and specific biochemical indicators with potential sex-related differences. T-test and chi-square test were performed for the statistical analyses. RESULTS: Of the 4,660 study participants, 10.26% had cholestasis; the prevalence of cholestasis increased with increasing age in male patients. The distribution of the cholestasis incidence according to the type of chronic liver disease was: 75.00%, primary sclerosing cholangitis; 42.86%, primary biliary cirrhosis; 35.97%, hepatic tumor; 30.77%, autoimmune hepatitis; 28.31%, drug-induced liver disease; 16.46%, alcoholic hepatitis; 13.98%, cryptogenic cirrhosis; 12.99%, schistosomal cirrhosis; 7.53%, alcoholic cirrhosis; 7.32%, mixed cirrhosis; 5.94%, viral liver cirrhosis; 2.70%, nonalcoholic fatty liver disease. There was no significant difference in the prevalence of cholestasis between the two sexes. In the patients with cholestasis, the levels of GGT and total bilirubin were significantly different between the two sexes. CONCLUSION: The incidence rate of cholestasis in first-hospitalized patients with chronic liver disease was 10.26%, and the rate increased with increased age. Patients with primary sclerosing cholangitis or primary biliary cirrhosis had higher incidence rates of cholestasis. Incidence rates of cholestasis of the various chronic liver diseases were not related to sex.

Associations between MTHFR Ala222Val polymorphism and risks of hepatitis and hepatitis-related liver cancer: a meta-analysis.

Chronic infection of viral hepatitis is the main cause of liver cancer. There were many studies assessing the associations of methylenetetrahydrofolate reductase (MTHFR) Ala222Val polymorphism with risks of hepatitis and hepatitis-related liver cancer, but no consistent results were reported. To investigate the associations of MTHFR Ala222Val polymorphism with risks of hepatitis and hepatitis-related liver cancer, we performed a meta-analysis of published case-control studies. Eligible studies were searched from PubMed and Chinese National Knowledge Infrastructure (CNKI) databases. The odds ratio (OR) and corresponding 95 % confidence interval (95 %CI) were used to assess the associations. Twenty-one individual studies with a total of 8,187 subjects were included. Overall, MTHFR Ala222Val polymorphism was not significantly associated with risks of liver cancer, hepatitis-related liver cancer, and non-hepatitis-related liver cancer. However, MTHFR Ala222Val polymorphism was significantly associated with risk of hepatitis infection (Val vs. Ala: OR = 1.15, 95 %CI 1.01-1.32, P = 0.03; ValVal/AlaVal vs. AlaAla: OR = 1.37, 95 %CI 1.11-1.68, P = 0.003). Therefore, MTHFR Ala222Val polymorphism is significantly associated with risk of hepatitis infection but not liver cancer. More studies are needed to further assess the association between MTHFR Ala222Val polymorphism and hepatitis-related liver cancer.

A matched case-control study of hepatitis B virus mutations in the preS and core promoter regions associated independently with hepatocellular carcinoma.

This study aimed to determine hepatitis B virus (HBV) mutations associated independently with the risk of hepatocellular carcinoma (HCC), as adjusted with other mutations in the preS and core promoter regions of HBV genotypes B and C. One hundred and forty HBV-infected patients with HCC and 280 HBV-infected patients without HCC who had intact data of HBV genotyping and DNA sequencing in both regions were involved in this age-, sex-matched case-control study. Univariate and two-step stepwise multivariate regression analyses were performed to determine factors associated with the risk of HCC. Of 39 mutations evaluated, 23 in genotype C and 6 in genotype B were associated with an increased risk of HCC in the univariate analysis. Multivariate analyses established that genotype C (adjusted odds ratio [AOR] = 3.3; 95% confidence interval [CI] = 1.1-9.8), viral load (≥10(4) copies/ml) (AOR = 2.4; 95% CI = 1.0-5.8), A2962G (AOR = 18.7; 95% CI = 7.5-46.7), preS2 start codon mutation (AOR = 12.5; 95% CI = 3.4-45.5), C105T (AOR = 0.1; 95% CI = 0.0-0.2), T1753V (AOR = 3.1; 95% CI = 1.1-9.2), and A1762T/G1764A (AOR = 2.9; 95% CI = 1.1-7.3) were associated independently with HCC, adjusted for factors including mutations in both regions. By using an estimating haplotype frequencies program, it was found that a haplotypic carriage with 105C and 2962G was significantly more frequent in the patients with HCC than in those without HCC and the frequency of haplotype 2962G-preS2 start codon wildtype-105C-1762T/1764A was 47.9% in the patients with HCC and 4.3% in those without HCC. Conclusively, A2962G and T105C are novel factors associated independently with HCC. Further prospective studies are needed to confirm the role of these mutations in the development of HCC.

Significant association of different preS mutations with hepatitis B-related cirrhosis or hepatocellular carcinoma.

BACKGROUND: The associations of nucleotide substitution mutations in the preS region of hepatitis B virus (HBV) with hepatocellular carcinoma (HCC) and cirrhosis remain unknown. We aimed to determine the associations of preS mutations with HCC or cirrhosis. METHODS: HBV from 603 asymptomatic hepatitis B surface antigen carriers (ASCs), 219 chronic hepatitis B (CHB) patients, 119 cirrhosis patients, and 231 HCC patients were genotyped and sequenced in the preS region. Nucleotides with the highest frequencies in HBV from the hepatitis B e antigen (HBeAg)-positive ASCs were treated as "wild-type" nucleotides. Twenty-one preS1 mutations and 14 preS2 mutations were evaluated. Multivariate regression analyses were applied to determine factors independently associated with cirrhosis or HCC. RESULTS: Most (85.7%) preS2 mutations were associated with CHB compared with ASCs, whereas most preS1 mutations were associated with HCC compared with the cirrhosis patients or CHB patients. Compared with the CHB patients, 81.0% preS1 mutations in genotype C were inversely associated with cirrhosis. Multivariate regression analyses showed that C2964A, C3116T, and C7A were novel factors associated with HCC compared with those without HCC, whereas A2964C and T3116C were independently associated with cirrhosis compared with ASCs and the CHB patients. Combined preS1 mutations had specificities greater than 95%, while C3116T and C7A had moderate sensitivities and specificities, for HCC. CONCLUSIONS: C2964A, C3116T, and C7A are novel markers independently associated with an increased risk of HCC, while A2964C and T3116C are novel markers independently associated with an increased risk of cirrhosis. Combined preS1 mutations are specific for HCC.

IkappaBalpha gene promoter polymorphisms are associated with hepatocarcinogenesis in patients infected with hepatitis B virus genotype C.

Genetic predisposition of nuclear factor-kappa B (NF-kappaB)-signaling pathways linking inflammation to hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) remains unresolved. We conducted a case-control study to determine the associations of the polymorphisms within the promoter regions of NFKB1 encoding NF-kappaB1 and NFKBIA encoding IkappaBalpha with the development of HCC. A total of 404 healthy controls, 482 non-HCC subjects with HBV infection and 202 patients with HCC were included. NFKB1 -94ATTG2 allele and GG allele in the 3'-untranslated region of NFKBIA were more prevalent in HCC patients than in the healthy controls. NFKBIA -826CT and NFKBIA -881AG allelic carriages were more prevalent in HCC patients than in the non-HCC subjects with HBV infection. The estimated haplotype frequency of NFKBIA promoter -881G-826T-519C was significantly higher in the patients with HCC than in the HBV-infected subjects without HCC (odds ratio = 3.142, P = 0.002). As compared with the HBV-infected subjects without HCC, NFKBIA -826 T and NFKBIA -881AG allelic carriages were only associated with HCC risk in the subjects with HBV genotype C. The association of NFKBIA -881AG allelic carriage with HCC risk was not affected by liver cirrhosis (LC) status, alanine aminotransferase level and hepatitis B e antigen status. By multivariate regression analysis, NFKB1 -94ATTG2, NFKBIA -826T, NFKBIA -881AG and HBV genotype C were independently associated with an increased risk of HCC. In conclusion, NFKB1 -94ATTG2 allele and haplotype -881G-826T-519C in NFKBIA promoter were associated with hepatocarcinogenesis. NFKBIA -826T and -881AG were associated with the risk of HCC in the subjects infected with HBV genotype C.

Role of hepatitis B virus genotype mixture, subgenotypes C2 and B2 on hepatocellular carcinoma: compared with chronic hepatitis B and asymptomatic carrier state in the same area.

The role of genotype mixture and subgenotypes remains controversial in determining the clinical outcome of chronic hepatitis B virus (HBV) infection. We aimed to determine their role on the development and the recurrence of hepatocellular carcinoma (HCC). HBV genotypes, serum viral load and hepatitis B e antigen (HBeAg) seroconversion were determined in 462 HCC patients, 234 chronic hepatitis patients and 425 asymptomatic carriers born in Eastern China. In the 462 HCC patients, 62 (13.4%), 337 (72.9%) and 49 (10.6%) had HBV subgenotype B2, C2 and genotype mixture, respectively. Genotype mixture in HCC patients and hepatitis patients was associated with higher viral load than HBV C2 (P = 0.012, P = 0.000) and more frequent than asymptomatic carriers (P = 0.005, P = 0.000). HBV C2 was more prevalent in HCC patients compared with controls. Proportion of HBV B2 in HCC patients decreased consecutively from <30 to 50-59 years group (P = 0.024). Age-related changes of HBeAg seroconversion were not consistent with serum viral load in HCC patients with HBV B2 and genotype mixture, quite in contrast to hepatitis patients. By multivariate regression analysis, age >or=40 years and serum viral load (>or=10 000 copies/ml) were independently associated with hepatocarcinogenesis, whereas age

Capsule oxymatrine in treatment of hepatic fibrosis due to chronic viral hepatitis: a randomized, double blind, placebo-controlled, multicenter clinical study.

AIM: To evaluate the efficacy and safety of oxymatrine capsule in treatment of hepatic fibrosis in patients with chronic viral hepatitis. METHODS: It was a randomized, double blind, placebo-controlled, multicenter clinical study. One hundred and forty-four patients were divided into oxymatrine capsule group(group A) and placebo group (group B). The course was 52 wk. Patients were visited once every 12 wk and the last visit was at 12 wk after cessation of the treatment. All patients had liver biopsy before treatment. part of them had a second biopsy at the end of therapy. Clinical symptoms, liver function test, serum markers of hepatic fibrosis were tested. Ultrasound evaluation was performed before, during and at the end of therapy. RESULTS: One hundred and forty-four patients enrolled in the study. Of them 132 patients completed the study according to the protocol,49 patients had liver biopsy twice (25 patients in group A and 24 in group B). At the end of therapy, significant improvements in hepatic fibrosis and inflammatory activity based on Semi-quantitative scoring system (SSS) were achieved in group A. The total effective rate of the treatment was 48.00%, much higher than that of 4.17% in group B (P<0.05). Significant improvement in serum markers of hepatic fibrosis such as hyaluronic acid (HA) and type III procollagenic peptide (P III P) in group A was seen (P<0.05). The total effective rate of serum markers at the end of therapy in group A was 68.19%, much higher than that of 34.85% in group B (P<0.05). The total effective rate of noninvasive markers at the end of therapy in group A was 66.67%, much higher than that of 30.30% in group B (P<0.05). The rate of adverse events was similar in two groups. CONCLUSION: Oxymatrine capsule is effective and safe in treatment of hepatic fibrosis due to chronic viral hepatitis.

Grading and staging of hepatic fibrosis, and its relationship with noninvasive diagnostic parameters.

AIM: To explore the grade and stage of pathology and the relationship between grading and staging of hepatic fibrosis and noninvasive diagnostic parameters. METHODS: Inflammatory activity and fibrosis of consecutive liver biopsies from 200 patients with chronic liver disease were determined according to the Diagnostic Criteria of Chronic Hepatitis in China, 1995. A comparative analysis was made in these patients comparing serum markers, Doppler ultrasonography, CT and/or MR imaging with the findings of liver biopsy. RESULTS: With increase of inflammatory activity, the degree of fibrosis also rose. There was a close correlation between liver fibrosis and inflammatory activity. AST, GGT, albumin, albumin/globulin, ALP, AFP, hyaluronic acid, N-terminal procollagen III(P III NP), collagen type IV(Col IV), tissue inhibitors of metalloproteinases-1 (TIMP-1), alpha-2-macroglobulin, natural killer cells(NK), some parameters of Doppler ultrasonography, CT and/or MR imaging were all related to the degree of inflammatory activity. GGT, albumin, albumin/globulin, ALP, AFP, hyaluronic acid, Col IV, TIMP-1, alpha-2- macroglobulin, transforming growth factor-beta 1 (TGFbeta1), NK, some parameters of Doppler ultrasonography, CT and/or MR imaging were all related to the staging of fibrosis. By regression analysis, the parameters used in combination to differentiate the presence or absence of fibrosis were age, GGT, the parameter of blood flow of portal vein per minute, the maximum oblique diameter of right liver by B ultrasound, the wavy hepatic surface contour by CT and/or MR. The sensitivity, specificity and accuracy of the above parameters were 80.36%, 86.67%, and 81.10%, respectively. CONCLUSION: There is close correlation between liver fibrosis and inflammatory activity. The grading and staging of liver fibrosis are related to serum markers, Doppler ultrasonography, CT and/or MR imaging. The combination of the above mentioned noninvasive parameters are quite sensitive and specific in the diagnosis of hepatic fibrosis.