Mengqi Zhu

Promoter Hypermethylation Mediated Downregulation of FBP1 in Human Hepatocellular Carcinoma and Colon Cancer

FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10) human hepatocellular carcinoma, 66.7% (6/9) liver cancer cell lines and 100% (6/6) colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2′-deoxycytidine (Aza), indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.

Acetylcarnitine Is a Candidate Diagnostic and Prognostic Biomarker of Hepatocellular Carcinoma

The identification of serum biomarkers to improve the diagnosis and prognosis of hepatocellular carcinoma has been elusive to date. In this study, we took a mass spectroscopic approach to characterize metabolic features of the liver in hepatocellular carcinoma patients to discover more sensitive and specific biomarkers for diagnosis and progression. Global metabolic profiling of 50 pairs of matched liver tissue samples from hepatocellular carcinoma patients was performed. A series of 62 metabolites were found to be altered significantly in liver tumors; however, levels of acetylcarnitine correlated most strongly with tumor grade and could discriminate between hepatocellular carcinoma tumors and matched normal tissues. Post hoc analysis to evaluate serum diagnosis and progression potential further confirmed the diagnostic capability of serum acetylcarnitine. Finally, an external validation in an independent batch of 58 serum samples (18 hepatocellular carcinoma patients, 20 liver cirrhosis patients, and 20 healthy individuals) verified that serum acetylcarnitine was a meaningful biomarker reflecting hepatocellular carcinoma diagnosis and progression. These findings present a strong new candidate biomarker for hepatocellular carcinoma with potentially significant diagnostic and prognostic capabilities. Cancer Res; 76(10); 2912-20. ©2016 AACR.

The role of plasma cell-free DNA detection in predicting preoperative chemoradiotherapy response in rectal cancer patients

In the present study, we studied the relationship between plasma cell-free DNA and the effect of preoperative chemoradiotherapy in patients with rectal cancer. The concentration, KRAS mutation and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status of cell-free DNA were measured by using polymerase chain reaction (PCR) analyses. The response to chemoradiotherapy was assessed using tumor regression grading (TRG) scores. The cell-free DNA concentrations in patients with rectal cancer (n=34) were significantly higher compared to healthy controls (n=10). The 400-base pair (bp) DNA concentration, 400-/100-bp DNA ratio decreased significantly after chemoradiotherapy in the good response group. The incidence of KRAS mutation decreased significantly after chemoradiotherapy in both good and poor response groups. Higher MGMT promoter methylation status at baseline DNA was associated with a better tumor response. Therefore, cell-free DNA detection may be useful in evaluating the effect of preoperative chemoradiotherapy in patients with rectal cancer.

The combination of three molecular markers can be a valuable predictive tool for the prognosis of hepatocellular carcinoma patients

Based on molecular profiling, several prognostic markers for HCC are also used in clinic, but only a few genes have been identified as useful. We collected 72 post-operative liver cancer tissue samples. Genes expression were tested by RT-PCR. Multilayer perceptron and discriminant analysis were built, and their ability to predict the prognosis of HCC patients were tested. Receiver operating characteristic (ROC) analysis was performed and multivariate analysis with Cox’s Proportional Hazard Model was used for confirming the markers’predictive efficiency for HCC patients’survival. A simple risk scoring system devised for further predicting the prognosis of liver tumor patients. Multilayer perceptron and discriminant analysis showed a very strong predictive value in evaluating liver cancer patients’prognosis. Cox multivariate regression analysis demonstrated that DUOX1, GLS2, FBP1 and age were independent risk factors for the prognosis of HCC patients after surgery. Finally, the risk scoring system revealed that patients whose total score >1 and >3 are more likely to relapse and die than patients whose total score ≤1 and ≤3. The three genes model proposed proved to be highly predictive of the HCC patients’ prognosis. Implementation of risk scoring system in clinical practice can help in evaluating survival of HCC patients after operation.

Increased activity of the complement system in cerebrospinal fluid of the patients with Non-HIV Cryptococcal meningitis

BackgroundsCryptococcal meningitis (CM) has been known to lead to significant morbidity and mortality. The relative contribution of the complement system in protection and pathogenesis during CM remains largely unknown. The purpose of this study was to evaluate the baseline complement component profiles in human cerebrospinal fluid (CSF) and plasma from non-HIV patients with CM, and therefore to provide insights of possible roles of the complement system in CM.MethodsCSF and blood samples from forty two CM patients not infected with HIV and thirteen non-CM control patients (Ctrl) were retrospectively selected and evaluated from the patients admitted to the hospital with a suspected diagnosis of CM. CSF and blood samples were collected at the admission. Enzyme-linked immunosorbent assay (ELISA) for complement components, cytokine IL-12 and western blot for C3 activation were performed on CSF and plasma samples. The levels of complement C1q, factor B (FB), mannose binding lectin (MBL), C2, C3, C4, C5, C4 binding protein (C4BP), Factor I (FI), Factor H (FH), sC5b-9 in CSF and plasma samples were compared. Pearson’s correlation coefficients were calculated on variables between complement components and the levels of total protein in the CSF samples.ResultsOur data demonstrated that the CSF levels of complement components of C1q, FB, MBL as well as complement pathway factors sC5b-9 and complement regulator FH were all elevated in patients with CM as compared to the controls, CSF C3 breakdown products iC3b were found in both CSF and plasma samples of the CM patients. A positive correlation was found between the levels of CSF protein and MBL, C1q or FB.ConclusionsThe activity of the complement system in CSF was increased in non-HIV patients with CM. C1q, MBL and FB are the important participants in the complement activation in CM. The relative contribution of each of the specific complement pathways and complement cascades in protection and inflammation resolution against CM warrant further investigation.

Serum MicroRNA Levels as a Noninvasive Diagnostic Biomarker for the Early Diagnosis of Hepatitis B Virus-Related Liver Fibrosis.

Background/Aims To investigate the role of selected serum microRNA (miRNA) levels as potential noninvasive biomarkers for differentiating S0–S2 (early fibrosis) from S3–S4 (late fibrosis) in patients with a chronic hepatitis B virus (HBV) infection. Methods One hundred twenty-three treatment-naive patients with a chronic HBV infection who underwent a liver biopsy were enrolled in this study. The levels of selected miRNAs were measured using a real-time quantitative polymerase chain reaction assay. A logistic regression analysis was performed to assess factors associated with fibrosis progression. Receiver operating characteristic (ROC) curve and discriminant analyses validated these the ability of these predicted variables to discriminate S0–S2 from S3–S4. Results Serum miR-29, miR-143, miR-223, miR-21, and miR-374 levels were significantly downregulated as fibrosis progressed from S0–S2 to S3–S4 (p<0.05), but not miR-16. The multivariate logistic regression analysis identified a panel of three miRNAs and platelets that were associated with a high diagnostic accuracy in discriminating S0–S2 from S3–S4, with an area under the curve of 0.936. Conclusions The levels of the studied miRNAs, with the exception of miR-16, varied with fibrosis progression. A panel was identified that was capable of discriminating S0–S2 from S3–S4, indicating that serum miRNA levels could serve as a potential noninvasive biomarker of fibrosis progression.

Role of interleukin-23 in monocyte-derived dendritic cells of HBV-related acute-on-chronic liver failure and its correlation with the severity of liver damage.

BACKGROUND The role of interleukin-23 (IL-23) in monocyte-derived dendritic cells (MoDCs) from hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) patients remains unclear. The aim of this study was to observe the correlation between the activation of the IL-23 signaling pathway and the prognosis of HBV-ACLF patients. MATERIALS AND METHODS The baseline levels of serum IL-6, IL-12, IL-17, IL-23, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) from immune tolerant (IT), chronic hepatitis B (CHB), HBV-ACLF patients and healthy individuals who served as healthy controls (HCs) were analyzed using the Luminex system, whereas serum IL-23 from HBV-ACLF patients was measured by ELISA before and after treatment. Purified monocytes were isolated from peripheral blood and were induced into MoDCs, IL-23, IL-23R, NF-κB and TRAF6 expression in MoDCs from 4 groups was analyzed using real-time PCR, and IL-23R and intracellular IL-23 were evaluated by flow cytometry. RESULTS Serum IL-6, IL-12, IL-17, IL-23 and TNF-α levels were upregulated in HBV-ACLF patients compared with IT patients, CHB patients and HCs (P<0.05 for all). Serum IL-23 was closely correlated with elevated serum IL-17 in HBV-ACLF patients (r=0.5935, P<0.0001). Moreover, IL-23 and IL-23R levels were significantly upregulated in MoDCs from patients with CHB or HBV-ACLF compared with HCs, and further upregulation of IL-23 and IL-23R was observed in HBV-ACLF patients compared to CHB patients (P<0.05 for all). IL-23 expression was markedly enhanced and was correlated with elevated NF-κB and TRAF6 in MoDCs from HBV-ACLF patients (P<0.05 for both). Linear correlation analysis demonstrated significant correlations between the expression of IL-23 and disease severity markers (MELD scoring system, international normalized ratio, prothrombin time and total bilirubin, r=0.6874, r=0.6475, r=0.6249, r=0.3771, respectively, P<0.05 for all) for individual HBV-ACLF patients, and IL-23 levels were significantly upregulated in non-survival HBV-ACLF patients compared with survival HBV-ACLF patients (P<0.05). CONCLUSION IL-23 in serum and MoDCs is significantly elevated in HBV-ACLF patients, TRAF6/NF-κB may play a role in IL-23 production by MoDCs in HBV-ACLF patients and high pre-treatment IL-23 levels in MoDCs are associated with mortality in HBV-ACLF patients.

Interleukin-23 mediates the pathogenesis of LPS/GalN-induced liver injury in mice

Background Interleukin‐23 (IL‐23) is required for T helper 17 (Th17) cell responses and IL‐17 production in hepatitis B virus infection. A previous study showed that the IL‐23/IL‐17 axis aggravates immune injury in patients with chronic hepatitis B virus infection. However, the role of IL‐23 in acute liver injury remains unclear. Objective The purpose of this study was to determine the role of the inflammatory cytokine IL‐23 in lipopolysaccharide/d‐galactosamine (LPS/GalN)‐induced acute liver injury in mice. Methods Serum IL‐23 from patients with chronic hepatitis B virus (CHB), acute‐on‐chronic liver failure (ACLF) and healthy individuals who served as healthy controls (HCs) was measured by ELISA. An IL‐23p19 neutralizing antibody or an IL‐23p40 neutralizing antibody was administered intravenously at the time of challenge with LPS (10 &mgr;g/kg) and GalN (400 mg/kg) in C57BL/6 mice. Hepatic pathology and the expression of Th17‐related cytokines, including IL‐17 and TNF‐&agr;; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and the stabilization factor Csf3 were assessed in liver tissue. Results Serum IL‐23 was significantly upregulated in ACLF patients compared with CHB patients and HCs (P < 0.05 for both). Serum IL‐23 was significantly upregulated in the non‐survival group compared with the survival group of ACLF patients, which was consistent with LPS/GalN‐induced acute hepatic injury in mice (P < 0.05 for both). Moreover, after treatment, serum IL‐23 was downregulated in the survival group of ACLF patients (P < 0.001). Compared with LPS/GalN mice, mice treated with either an IL‐23p19 neutralizing antibody or an IL‐23p40 neutralizing antibody showed less severe liver tissue histopathology and significant reductions in the expression of Th17‐related inflammatory cytokine, including IL‐17 and TNF‐&agr;; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and stabilization factors Csf3 within the liver tissue compared with LPS/GalN mice (P < 0.05 for all). Conclusion High serum IL‐23 was associated with mortality in ACLF patients and LPS/GalN‐induced acute liver injury in mice. IL‐23 neutralizing antibodies attenuated liver injury by reducing the expression of Th17‐related inflammatory cytokines, neutrophil chemoattractants and stabilization factors within the liver tissue, which indicated that IL‐23 likely functions upstream of Th17‐related cytokine and chemokine expression to recruit inflammatory cells into the liver. HighlightsIL‐23 was associated with mortality in acute on chronic liver failure patients.IL‐23 neutralizing antibodies attenuated LPS/GalN‐induced acute liver injury in mice.The mechanism of attenuating liver injury involves regulation of Th17‐related cytokine and chemokine expression in liver.

MicroRNA-548j inhibits type I interferon production by targeting ZBTB11 in patients with chronic hepatitis B

Type I IFNs play crucial roles in antiviral immune responses. By inducing cellular resistance to viral infection and apoptosis of virus-infected cells, they impair virus replication and eliminate the invading pathogens. However, in CHB patients, generation of type I IFN was significantly impaired and the underlying mechanisms have not been fully understood. MicroRNA-548 family has been implicated in regulating antiviral response upon various infections. Here we explored the potential role of miR-548j in modulating type I IFN production in CHB. We found that miR-548j expression increased in MoDCs from CHB patients than that from healthy volunteers and transient transfection of miR-548j mimic led to a marked decrease of IFN-α/β production in MoDCs from healthy volunteers while blockade of miR-548j by anti-miR-548j significantly restored IFN-α/β secretion in MoDCs from CHB patients. In addition, Zinc finger and BTB domain containing 11 (ZBTB11) was predicted and finally confirmed to be a target of miR-548j. Forced expression of ZBTB11 also restored IFN-α/β secretion in MoDCs from CHB patients. These results indicate the involvement of miR-548j in aberrant production of type I IFN in CHB patients, and provide a potential target for developing anti-HBV therapies.

Factors associated with significant liver necroinflammation in chronic hepatitis B patients with cirrhosis

We determined the association between various clinical parameters and significant liver necroinflammation in patients with chronic hepatitis B (CHB) related cirrhosis. Two hundred patients with CHB related cirrhosis were recruited in the final analysis. Clinical laboratory values and characteristics were obtained from the medical record. We performed analyses of the relationships between independent variables and significant liver necroinflammation by using binary logistic regression analysis and discriminant analysis. Significant liver necroinflammation (grade≥2) was found in 58.0% (80/138) of antiviral therapy patients and 48.4% (30/62) of non antiviral therapy patients respectively. Also, there were some significant differences in serum hepatitis B surface antigen (HBsAg), serum hepatitis B e antigen (HBeAg) and serum hepatitis B virus (HBV) DNA between antiviral therapy and non antiviral therapy patients. After that, aspartate aminotransferase (AST), total bilirubin (TBIL), total bile acid (TBA), prothrombin time (PT), aspartate aminotransferase to platelet ratio index (APRI) and serum HBV DNA were confirmed as independent predictors of significant liver necroinflammation in CHB patients with cirrhosis by univariate analysis and multivariate analysis (p = 0.002, 0.044, 0.001, 0.014, 0.01 and 0.02 respectively). Finally, receiver operating characteristic (ROC) curve analysis and discriminant analysis validated that these six variables together have strong predictive power to evaluate significant liver necroinflammation.