MiRNA-548ah, a potential molecule associated with transition from immune tolerance to immune activation of chronic hepatitis B.

OBJECTIVE: The present study aims to identify the differently expressed microRNA (miRNA) molecules and target genes of miRNA in the immune tolerance (IT) and immune activation (IA) stages of chronic hepatitis B (CHB). METHODS: miRNA expression profiles of peripheral blood mononuclear cells (PBMCs) at the IT and IA stages of CHB were screened using miRNA microarrays and authenticated using a quantitative real-time polymerase chain reaction (RT-PCR). Gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) were used to analyze the significant functions and pathways of possible target genes of miRNAs. Assays of the gain and loss of function of the miRNAs were performed to verify the target genes in THP-1 cell lines. The luciferase reporter test was used on 293T cells as direct targets. RESULTS: Significantly upregulated miR-548 and miR-4804 were observed in the miRNA microarrays and confirmed by RT-PCR in PBMCs at the IT and IA stages of CHB. GO and KEGG analysis revealed that MiR-548 and miR-4804 could be involved in numerous signaling pathways and protein binding activity. IFNγR1 was predicted as a target gene and validated as the direct gene of MiR-548. Significant negative correlation was found between the miR-548ah and mRNA levels of IFN-γR1 in CHB patients. CONCLUSIONS: The abnormal expression profiles of miRNA in PBMCs could be closely associated with immune activation of chronic HBV infection. miR-548, by targeting IFN-γR1, may represent a mechanism that can facilitate viral pathogenesis and help determine new therapeutic molecular targets.

Screening and bioinformatic analysis of microRNA-associated immune clearance in patients with chronic hepatitis B.

OBJECTIVE: This study aimed to perform screening and bioinformatic analysis of microRNA (miRNA) molecules associated with immune clearance in chronic hepatitis B (CHB) patients. METHODS: Peripheral blood mononuclear cells (PBMCs) of CHB patients and healthy individuals were collected and detected by microarray. The target genes of differentially expressed miRNA molecules were predicted using three databases. Their molecular pathways and functions were analyzed by bioinformatics methods. RESULTS: Compared with healthy individuals, 52 differentially expressed miRNA molecules were found in PBMCs of CHB patients, of which 33 were up-regulated and 19 were down-regulated. A total of 354 target genes were predicted in up-regulated miRNA molecules, and 1935 target genes were predicted in down-regulated miRNA molecules. MiRNA-mRNA network analysis showed that some target genes might be regulated, and constituted complex molecular networks with hsa-miR-520d-5p, hsa-miR-106a-5p, hsa-miR-30a-5p, and hsa-miR-29b-3p. Gene ontology and pathway analyses showed that several molecular pathways might be affected by up- or down-regulated miRNA molecules. CONCLUSION: Abnormal expression of multiple miRNA molecules in PBMCs of CHB patients might be involved in immune clearance pathogenesis through the regulation of multiple molecular pathways and target genes.

Mechanism and efficacy of mobilization of granulocyte colony-stimulating factor in the treatment of chronic hepatic failure.

BACKGROUND/AIMS: To explore the efficacy of G-CSF mobilization in the treatment of chronic liver failure (CLF) and the mechanism of its action. METHODOLOGY: The proportions of cluster-of-differentiation (CD)-34+ cells and their receptor-CXCR4 were detected by flow cytometry in patients with different types of chronic HBV infection. The levels of chemokines and cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: The proportion of CD34+ cells in patients with cirrhosis was significantly increased compared with the healthy controls (p<0.05) and was increased obviously after treatment by G-CSF mobilization (p<0.01). The expression levels of SDF-1, SCF and MMP-9 were significantly elevated in patients with chronic hepatitis B and liver cirrhosis (p<0.01). The expression levels of SCF and MMP-9 were significantly elevated after treatment with G-CSF (p<0.05). No significant differences were found in the levels of total bilirubin, albumin and prothrombin time between the treated and control groups; furthermore, no significant differences were observed in the cure and improvement rates between the two groups. CONCLUSIONS: The basal levels of stem cell mobilization in patients with liver cirrhosis might be associated with the repair of liver injury. G-CSF could promote hematopoietic stem cell mobilization through regulation of the expression levels of stem-cell-mobilization-related factors in patients with liver cirrhosis. No apparent effects of G-CSF therapy on both liver function and short-term prognosis in patients with liver cirrhosis were confirmed.

Association of T regulatory cells with natural course and response to treatment with interferon-α in patients with chronic hepatitis B infection.

BACKGROUND: Regulatory T cell populations, particularly CD4(+)CD25(+) T regulatory cells, have been implicated in the persistence of hepatitis B virus (HBV) infection. However, no clear relationship has been established between the frequency of CD4(+)CD25(+) T regulatory cells in the peripheral blood and either the disease phases in the natural history of chronic HBV infection or in the response to interferon-α therapy. METHODS: In the present study, three different common markers of CD4(+)CD25(+) T regulatory cells were used to determine the numbers of T regulatory cells in healthy controls and in patients with chronic HBV infection. RESULTS: No significant difference was found when samples were gated for CD25(hi) and CD25(+)FoxP3(+) T cells. A significant correlation was found between the number of CD4(+) Treg cells that gated with CD25(+)FoxP3(+) and CD25(+)CD127(low/-) in healthy controls and in patients with chronic hepatitis B (CHB) (r = 0.67, 0.59; P < 0.01). The percentages of Treg cells were (8.56 ± 2.01)% in asymptomatic carriers (Asc), (8.74 ± 3.04)% in inactive HBsAg carriers, (10.7 ± 2.93)% in CHB and (7.42 ± 1.28)% in healthy controls (F = 11.1, P < 0.001). The percentage of Treg cells in patients with CHB was higher than in asymptomatic HBV patients, inactive HBsAg carriers, or healthy controls (P < 0.01). The proportion of CD4(+)CD25(+)CD127(low/-) T cells in patients who responded to interferon-α was (11.9 ± 3.3)%, (9.1 ± 2.4)% and (9.0 ± 2.9)% at baseline, week 12 and week 24 after treatment, respectively (Z = 2.42, P < 0.05; Z = 2.67, P < 0.01). CONCLUSIONS: These results suggest that the proportion of the CD4(+)CD25(+) regulatory T cells might be affected by the application of different markers in process to detect T regulatory cells. The frequency of Treg cells was increased in patients with CHB, which might be associated with the disease activity of these patients and contribute to prevention of extensive liver damage. A decline in Treg cells at week 12 of treatment might be associated with a better response to treatment with interferon-α.

Application of interferon response-related gene array to the antiviral treatment outcome in chronic hepatitis C.

BACKGROUND/AIMS: It has been known that viral factors such as genotype and viral load have a major influence on the outcome of antiviral treatment. Nevertheless, researchers have become increasingly aware that host genetic factors can modulate the response to antiviral treatment. The underlying mechanisms for the varying virologic response rates to IFNalpha-based antiviral therapy are unknown. METHODOLOGY: RNA was isolated from peripheral blood monocytes from treatment-naïve patients with chronic hepatitis C before and at the end of treatment. Gene expression was measured using SuperArray microarrays and compared to that of healthy controls. RESULTS: Ten patients were classified as rapid responders (RRs) and seven patients as non-RRs according to the serum HCV RNA level after 4 weeks of treatment in 17 patients with CHC. Compared with healthy controls, nine and eighteen different expression genes were found significantly in patients with RR and N-RR, respectively. Five different expression genes were found between the patients with RR and N-RR. Two genes that were down-regulated were found between HCV genotype 1b and genotype 2a. Seven different expression genes that were all down-regulated were found between the patients with ETVR and N-ETVR. CONCLUSIONS: (1) The down-regulation of some IFN response-related genes are associated with null response to treat with interferon. (2) It should be HCV genotype 1b is more successful in inducing the down-regulation of IFN response-related genes than HCV genotype 2a, thus contribute to the resistance to IFN.

Preliminary identification and analysis of point mutations correlated with response to interferon-alpha in hepatitis B virus post-transcriptional regulatory elements.

BACKGROUND: It is still unclear whether viral genetic variability influences response to interferon (IFN)-alpha treatment. Recent reports suggest that IFN-alpha effects may be associated with hepatitis B virus (HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-transcriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-alpha treatment. METHODS: The HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and comparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into HepG2 cells, which were then exposed to recombinant human cytokines. RESULTS: The T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patients. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-alpha/gamma and tumor necrosis factor (TNF)-alpha except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-alpha on HPRE. CONCLUSIONS: There are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-alpha. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-alpha other than that of IFN-gamma or TNF-alpha on HPRE.

Preliminary identification and analysis of different points related with response to interferon-alpha in HBV post-transcriptional regulatory element.

OBJECTIVE: To screen the differential points related with response to interferon-alpha in HBV post-transcriptional regulatory element (HPRE). METHODS: HPRE sequence of 31 Chinese patients with HBV infection were detected by direct sequencing of PCR products. Differential points in HPRE between Chinese patients and White patients were compared. RESULTS: The T to C differential point at nt 1 504 and C to T(G) at nt 1 508 were found respectively in HPRE of 21 and 19 patients with chronic hepatitis B, the C to T differential point at nt 1 509 were found in 17 cases. These differential points were not found in HPRE of the White patients. The relationship between the HPRE differential points and the levels of HBV DNA was not observed. CONCLUSION: There were differential points in the HPRE between Chinese and White HBV patients, which might be correlated with response to interferon-alpha.