Fengmin Lu

Aberrant expression of microRNA 155 may accelerate cell proliferation by targeting sex-determining region Y box 6 in hepatocellular carcinoma.

Recent research has suggested that the oncomir microRNA 155 (miR‐155) is up‐regulated in hepatocellular carcinoma (HCC). In this study, the authors investigated the tumorigenic mechanism of this oncomir in the development of HCC.

SOX17 antagonizes WNT/β-catenin signaling pathway in hepatocellular carcinoma

SRY-box containing gene 17 (SOX17) was reported to be indispensable for embryonic development and a candidate tumor suppressor gene which antagonizes the canonical WNT/β-catenin signaling pathway in colorectal cancer. In this study, we investigated the function and epigenetic regulation of SOX17 in human hepatocellular carcinoma (HCC). DNA methylation of SOX17 was analyzed in 62 human HCC tissues and HCC cell lines by MSP. A role as a tumor suppressor gene was evaluated by colony formation assay and regulation of WNT/β-catenin signal pathway by SOX17 was determined by IHC and luciferase reporter assay. DNA methylation of the SOX17 promoter region occurs in 82% of HCC tissues and is associated with nuclear accumulation of β-catenin. Restoration of SOX17 inhibits HepG2 colony formation and β-catenin/TCF-dependent transcription with the presence of HMG box in SOX17. In conclusion, SOX17 negatively regulates canonical WNT/β-catenin signaling pathway, and inhibits human HCC cells growth, providing an explanation for the loss of expression by epigenetic mechanisms in these tumors.

Transmission of hepatitis E virus from rabbits to cynomolgus macaques.

The recent discovery of hepatitis E virus (HEV) strains in rabbits in the People’s Republic of China and the United States revealed that rabbits are another noteworthy reservoir of HEV. However, whether HEV from rabbits can infect humans is unclear. To study the zoonotic potential for and pathogenesis of rabbit HEV, we infected 2 cynomolgus macaques and 2 rabbits with an HEV strain from rabbits in China. Typical hepatitis developed in both monkeys; they exhibited elevated liver enzymes, viremia, virus shedding in fecal specimens, and seroconversion. Comparison of the complete genome sequence of HEV passed in the macaques with that of the inoculum showed 99.8% nucleotide identity. Rabbit HEV RNA (positive- and negative-stranded) was detectable in various tissues from the experimentally infected rabbits, indicating that extrahepatic replication may be common. Thus, HEV is transmissible from rabbits to cynomolgus macaques, which suggests that rabbits may be a new source of human HEV infection.

Phylogenetic analysis of 626 hepatitis E virus (HEV) isolates from humans and animals in China (1986-2011) showing genotype diversity and zoonotic transmission.

Hepatitis E is considered as a public health problem in China. To determine the overall molecular epidemiology of hepatitis E virus (HEV) and analyze the situation of cross-species transmission between humans and swine in China over the last 25 years (1986-2011), 626 HEV complete and partial sequences (89 isolates identified by our group) isolated from humans and animals in China were retrieved from GenBank and subjected to phylogenetic analysis. There were three genotypes and 11 sub-genotypes of HEV prevailing in China. Furthermore, rabbit HEVs, of which the genotype is controversial, are also widespread in China. Genotype 1 was the most isolated genotype prior to 2000 and mainly detected in Xinjiang, Beijing and East China. However, genotype 4, which was identified in most regions of China during the last 10 years, has overtaken genotype 1 in frequency of isolation nationwide. Genotype 3 HEV strains have been found only in eastern China and were thought to be imported from Japan. Both genotypes 3 and 4 were found in humans and swine and cross-species transmission from pigs to humans of the two genotypes may have occurred in Northeast, Northwest, North, East and South China. These results indicate that HEV strains with considerable genetic diversity are widespread and the zoonotic transmission between swine and humans appears ubiquitous in China.

Epidemiology and prevention of hepatitis B virus infection in China

Summary.  The concept of a hepatitis B vaccine was first introduced into China in 1978. China has been one of the first two developing countries to enact the universal hepatitis B vaccination programme for newborn babies in 1992, and has made tremendous achievements in the control of hepatitis B virus (HBV) infection since then. China now has both low and high endemic regions regarding HBV prevalence co‐existing. Although China’s drive to stop HBV spread has resulted in changes in HBV epidemic patterns, for the eventual elimination HBV infection in China, it is important to understand the current status of its epidemiology and the aspects of HBV transmission in different regions. More efforts are needed to improve and develop strategies for the control of HBV infection in China, particularly after implementing the policy of universal HBV immunization for all newborns.

Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication.

AIM To screen and investigate the effective gRNAs against hepatitis B virus (HBV) of genotypes A-D. METHODS A total of 15 gRNAs against HBV of genotypes A-D were designed. Eleven combinations of two above gRNAs (dual-gRNAs) covering the regulatory region of HBV were chosen. The efficiency of each gRNA and 11 dual-gRNAs on the suppression of HBV (genotypes A-D) replication was examined by the measurement of HBV surface antigen (HBsAg) or e antigen (HBeAg) in the culture supernatant. The destruction of HBV-expressing vector was examined in HuH7 cells co-transfected with dual-gRNAs and HBV-expressing vector using polymerase chain reaction (PCR) and sequencing method, and the destruction of cccDNA was examined in HepAD38 cells using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these gRNAs was assessed by a mitochondrial tetrazolium assay. RESULTS All of gRNAs could significantly reduce HBsAg or HBeAg production in the culture supernatant, which was dependent on the region in which gRNA against. All of dual gRNAs could efficiently suppress HBsAg and/or HBeAg production for HBV of genotypes A-D, and the efficacy of dual gRNAs in suppressing HBsAg and/or HBeAg production was significantly increased when compared to the single gRNA used alone. Furthermore, by PCR direct sequencing we confirmed that these dual gRNAs could specifically destroy HBV expressing template by removing the fragment between the cleavage sites of the two used gRNAs. Most importantly, gRNA-5 and gRNA-12 combination not only could efficiently suppressing HBsAg and/or HBeAg production, but also destroy the cccDNA reservoirs in HepAD38 cells. CONCLUSION These results suggested that CRISPR/Cas9 system could efficiently destroy HBV expressing templates (genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV cccDNA in chronic HBV infection patients.

Synthesis and characterization of chitosan-based hydrogels.

Biocompatible hydrogels based on water-soluble chitosan-ethylene glycol acrylate methacrylate (CS-EGAMA) and polyethylene glycol diamethacrylate (PEGDMA) were synthesized by photopolymerization. Characterization of morphology, weight loss, water state of hydrogel, pH-sensitivity and cytotoxicity were investigated by scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), X-ray diffraction (XRD), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR), swelling test and methylthiazolydiphenyl-tetrazolium bromide (MTT) assay. The results indicated that the hydrogels were sensitive to pH of the medium, no cytotoxicity for L929 and SW1353, satisfactory for the composite to be used in bioapplications.

Methylation-mediated repression of microRNA 129-2 enhances oncogenic SOX4 expression in HCC

Aberration of miR‐129‐2 has been linked to a variety of human tumours. However, whether miR‐129‐2 is involved in hepatocarcinogenesis remains unknown. Here, we investigate the involvement of miR‐129‐2 in HBV infection‐related HCC.

Re-evaluation of the Carcinogenic Significance of Hepatitis B Virus Integration in Hepatocarcinogenesis

To examine the role of hepatitis B virus (HBV) integration in hepatocarcinogenesis, a systematic comparative study of both tumor and their corresponding non-tumor derived tissue has been conducted in a cohort of 60 HBV associated hepatocellular carcinoma (HCC) patients. By using Alu-polymerase chain reaction (PCR) and ligation-mediated PCR, 233 viral-host junctions mapped across all human chromosomes at random, no difference between tumor and non-tumor tissue was observed, with the exception of fragile sites (P = 0.0070). HBV insertions in close proximity to cancer related genes such as hTERT were found in this study, however overall they were rare events. No direct correlation between chromosome aberrations and the number of HBV integration events was found using a sensitive array-based comparative genomic hybridization (aCGH) assay. However, a positive correlation was observed between the status of several tumor suppressor genes (TP53, RB1, CDNK2A and TP73) and the number of chromosome aberrations (r = 0.6625, P = 0.0003). Examination of the viral genome revealed that 43% of inserts were in the preC/C region and 57% were in the HBV X gene. Strikingly, approximately 24% of the integrations examined had a breakpoint in a short 15 nt viral genome region (1820–1834 nt). As a consequence, all of the confirmed X gene insertions were C-terminal truncated, losing their growth-suppressive domain. However, the same pattern of X gene C-terminal truncation was found in both tumor and non-tumor derived samples. Furthermore, the integrated viral sequences in both groups had a similar low frequency of C1653T, T1753V and A1762T/G1764A mutations. The frequency and patterns of HBV insertions were similar between tumor and their adjacent non-tumor samples indicating that the majority of HBV DNA integration events are not associated with hepatocarcinogenesis.

Baseline quantitative hepatitis B core antibody titre alone strongly predicts HBeAg seroconversion across chronic hepatitis B patients treated with peginterferon or nucleos(t)ide analogues

Objective The investigation regarding the clinical significance of quantitative hepatitis B core antibody (anti-HBc) during chronic hepatitis B (CHB) treatment is limited. The aim of this study was to determine the performance of anti-HBc as a predictor for hepatitis B e antigen (HBeAg) seroconversion in HBeAg-positive CHB patients treated with peginterferon (Peg-IFN) or nucleos(t)ide analogues (NUCs), respectively. Design This was a retrospective cohort study consisting of 231 and 560 patients enrolled in two phase IV, multicentre, randomised, controlled trials treated with Peg-IFN or NUC-based therapy for up to 2 years, respectively. Quantitative anti-HBc evaluation was conducted for all the available samples in the two trials by using a newly developed double-sandwich anti-HBc immunoassay. Results At the end of trials, 99 (42.9%) and 137 (24.5%) patients achieved HBeAg seroconversion in the Peg-IFN and NUC cohorts, respectively. We defined 4.4 log10 IU/mL, with a maximum sum of sensitivity and specificity, as the optimal cut-off value of baseline anti-HBc level to predict HBeAg seroconversion for both Peg-IFN and NUC. Patients with baseline anti-HBc ≥4.4 log10 IU/mL and baseline HBV DNA <9 log10 copies/mL had 65.8% (50/76) and 37.1% (52/140) rates of HBeAg seroconversion in the Peg-IFN and NUC cohorts, respectively. In pooled analysis, other than treatment strategy, the baseline anti-HBc level was the best independent predictor for HBeAg seroconversion (OR 2.178; 95% CI 1.577 to 3.009; p<0.001). Conclusions Baseline anti-HBc titre is a useful predictor of Peg-IFN and NUC therapy efficacy in HBeAg-positive CHB patients, which could be used for optimising the antiviral therapy of CHB.