Jinrong Zhao

HBc binds to the CpG islands of HBV cccDNA and promotes an epigenetic permissive state.

HBV covalently closed circular DNA (cccDNA) is the template for the transcription of HBV. HBV core protein (HBc) is a main component of the HBV cccDNA minichromosome. However, the function of HBc in cccDNA is not fully understood. In light of recent findings that HBV cccDNA may be regulated epigenetically, we analyzed the binding of HBc to cccDNA and the impact of HBc on cccDNA epigenetic profile in the liver biopsy samples of 22 patients with chronic Hepatitis B (CHB). We found that HBc binding to HBV cccDNA occurred preferentially at CpG island 2, an important region for the regulation of HBV transcription. Furthermore, the relative abundances of HBc binding to CpG island 2 were positively correlated with the ratios of relaxed circular DNA to cccDNA and the levels of serum HBV DNA in those patients. Interestingly, the relative abundances of HBc binding to CpG island 2 were associated with the binding of CREB binding protein (CBP) and with hypomethylation in CpG island 2 of HBV cccDNA minichromosomes. However, relatively higher amounts of HBc binding to CpG island 2 of cccDNA were accompanied by lower amounts of HDAC1 binding. Multivariate analysis revealed that the abundances of HBc binding to CpG island 2 of cccDNA and positive HBeAg were independent factors associated with the replication of HBV (p = 0.001 for both). Apparently, HBc is a positive regulator of HBV transcription and replication, maintaining the permissive epigenetic state in the critical region of the HBV cccDNA minichromosomes.

Hepatitis B viral core protein disrupts human host gene expression by binding to promoter regions

BackgroundThe core protein (HBc) of hepatitis B virus (HBV) has been implicated in the malignant transformation of chronically-infected hepatocytes and displays pleiotropic functions, including RNA- and DNA-binding activities. However, the mechanism by which HBc interacts with the human genome to exert effects on hepatocyte function remains unknown. This study investigated the distribution of HBc binding to promoters in the human genome and evaluated its effects on the related genes’ expression.ResultsWhole-genome chromatin immunoprecipitation microarray (ChIP-on-chip) analysis was used to identify HBc-bound human gene promoters. Gene Ontology and pathway analyses were performed on related genes. The quantitative polymerase chain reaction assay was used to verify ChIP-on-chip results. Five novel genes were selected for luciferase reporter assay evaluation to assess the influence of HBc promoter binding. The HBc antibody immunoprecipitated approximately 3100 human gene promoters. Among these, 1993 are associated with known biological processes, and 2208 regulate genes with defined molecular functions. In total, 1286 of the related genes mediate primary metabolic processes, and 1398 encode proteins with binding activity. Sixty-four of the promoters regulate genes related to the mitogen-activated protein kinase (MAPK) pathways, and 41 regulate Wnt/beta-catenin pathway genes. The reporter gene assay indicated that HBc binding up-regulates proto-oncogene tyrosine-protein kinase (SRC), type 1 insulin-like growth factor receptor (IGF1R), and neurotrophic tyrosine kinase receptor 2 (NTRK2), and down-regulates v-Ha-ras Harvey rat sarcoma viral oncogene (HRAS).ConclusionHBc has the ability to bind a large number of human gene promoters, and can disrupt normal host gene expression. Manipulation of the transcriptional profile in HBV-infected hepatocytes may represent a key pathogenic mechanism of HBV infection.

Soluble MHC I and Soluble MIC Molecules: Potential Therapeutic Targets for Cancer

It has become clear that soluble MHC I (sMHC I) and soluble MIC (sMIC), which are highly elevated in sera of cancer patients, can be viewed to be tolerogenic, and that metalloproteinases are involved in their generation process. In this review, an overview is provided of the recent progress made in the sMHC I and sMIC fields, with emphasis on their structure, formation, and function, and the key-questions that still await answers are addressed. Understanding better their formation mechanism, it will become more feasible to modulate the immune responses in cancer patients by targeting molecules involved in their generation process.

O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation Detection in Glioma Tumors by a Novel Fluorescence Polarization Assay

O6-Methylguanine-DNA methyltransferase methylation status has a very good predictive value for benefit from alkylating agent therapy. The stratified therapy assignment of patients according to the O6-Methylguanine-DNA methyltransferase methylation status requires a standardized diagnostic test. A novel method detecting the promoter methylation status of O6-Methylguanine-DNA methyltransferase in tissue samples by a fluorescence polarization assay was developed. A pair of primers was used to amplify a 266 bp fragment in the promoter region of the O6-Methylguanine-DNA methyltransferase gene. Two probes specific for either methylated or unmethylated DNA labeled with different fluorophores hybridized with their target amplicons, and the hybridization increased the fluorescence polarization values. The methylation status was determined by the increased fluorescence polarization values. Ninety-seven glioma tumor samples were analyzed in parallel with the new assay and the nested gel-based methylation-specific polymerase chain reaction assay. The results of the methylation status of the fluorescence polarization assay were in good concordance with the results obtained with the nested gel-based methylation-specific polymerase chain reaction assay. The sensitivity and stability of the fluorescence polarization assay have been measured. The coefficient of variation of the reproducibility for the fluorescence polarization assay was <10%. The minimum detection level established with the fluorescence polarization assay was 20 copies/&mgr;L. The fluorescence polarization assay allowed the discrimination of the O6-Methylguanine-DNA methyltransferase methylation status at individual CpG sites directly in the solution without the 2-step approach with nested primers.

The Influence of PSCA Gene Variation on Its Expression and Gastric Adenocarcinoma Susceptibility in the Northwest Chinese Population

Gastric adenocarcinoma (GAC) imposes a considerable health burden around the world. Gene variation in prostate stem cell antigen gene (PSCA) has been identified to be associated with GAC risk, while the results showed regional variation. To explore the influence of PSCA gene variation on its expression and GAC risk in the Northwest Chinese population, four single nucleotide polymorphisms (SNPs) of PSCA were genotyped in 476 GAC cases and 481 controls using MassARRAY system. Two SNPs of rs2294008 (C>T) and rs2976392 (G>A) were identified to be associated with GAC risk. rs2294008, rs2976392 and rs10216533 made up two statistically significant haplotypes (Hap-CGG and Hap-TAG). Additionally, PSCA expression was analyzed by quantitative real time PCR, immunohistochemistry and tissue microarray. The results showed that PSCA expression was decreased in GAC tissues compared with adjacent normal tissues. For normal tissues, PSCA expression was higher with Hap-TA than that with Hap-CG. For GAC tissues, the differentiation degree of Hap-TA was higher than that of Hap-CG. The expression distribution of PSCA in multiple human organs showed disparity. These results suggest that PSCA gene variation has a potential effect on its expression and GAC risk in the Northwest Chinese population.

A novel combined capillary chip for rapid identification of gene mutation

To develop a simple micro-platform for gene mutation detection, we used a DNA hybridization approach in a combined microchannel. A glass groove immobilized with oligonucleotide probes by UV-crosslinking, and encased in a transparent heat-shrinkable polythene tube. After heat treatment, the polythene tube stretched tightly over the glass groove and was designated as a combined capillary chip (CCC). The CCC assay was optimized with 10 μL reaction solutions shuttling back and forth at 50 μL min−1 for 10 min, give a detection minimum of 0.1 nM target DNA sequences. In hepatitis B virus (HBV) YMDD mutations detection, 61.3% (92/150) was of a single genotype and it was authenticated with sequence analysis. The other 38.7% (58/150) was mixed genotype detected, but 18.9% (11/58) has a missed diagnosis in sequence analysis. It proved a higher sensitivity than sequence analysis. The CCC assay has a simple fabricating process, simple structure and higher specificity in gene mutation detection. These features are promising for clinical gene mutation analysis.