Risk factors of low-level viremia in chronic hepatitis B patients receiving Entecavir monotherapy: a retrospective cohort study.

BACKGROUND AND AIM: Low-level viremia (LLV), a special case of poor response to antiviral therapy, has become a focus of liver disease research; however, most studies have focused on poor response to antiviral therapy, and little attention has been paid to LLV. Therefore, this study aimed to investigate the factors influencing LLV in patients with chronic hepatitis B (CHB) receiving entecavir (ETV) monotherapy. METHODS: Clinical data of CHB patients receiving ETV treatment for at least 1 year at the outpatient department of the Affiliated Hospital of Xuzhou Medical University from November 2018 to June 2020 were collected. Patients were divided into LLV (180 cases) and sustained virological response (SVR) groups (337 cases) according to the hepatitis B virus (HBV) DNA load at the end of the observation period. Demographic features and laboratory markers were also examined. Univariate and multivariate logistic regression analyses were performed to examine factors influencing LLV in patients receiving long-term ETV monotherapy. RESULTS: Significant differences were noted between the LLV and SVR groups in terms of age, sex, presence or absence of cirrhosis, HBeAg positivity rate, baseline HBV DNA load, and baseline HBsAg level before treatment. Multivariate logistic regression analysis showed that baseline HBeAg status, HBV DNA load, and HBsAg quantification were pretreatment risk factors for LLV in long-term ETV antiviral therapy. CONCLUSIONS: CHB patients with a high HBV DNA load, high HBsAg quantification, and positive HBeAg results tend to have a high risk of LLV despite long-term ETV antiviral treatment and should be dynamically monitored.

VDR rs7975232/ApaI genetic variation predicts sustained HBsAg loss in HBeAg-positive chronic hepatitis B patients treated with pegylated interferon.

AIM: To evaluate the predictive value of vitamin D and its metabolic pathway gene polymorphisms in response to pegylated interferon (Peg-IFN) in hepatitis B early antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. METHODS: One hundred and nineteen HBeAg-positive CHB patients who received Peg-IFN monotherapy for 48 weeks and then were followed-up for another 48 weeks were prospectively enrolled; baseline 25-hydroxy vitamin D (25-(OH)D) and hepatitis B virus serologic marker levels were detected, nine critical single nucleotide polymorphisms within vitamin D metabolism were genotyped. RESULTS: Forty-five (37.8%), 44 (37.0%), 35 (29.4%), and 11 (9.2%) of the patients achieved virological response (VR), HBeAg loss, combined response (CR), and hepatitis B surface antigen (HBsAg) level < 200 IU/mL at the end of treatment (EOT; week 48), respectively; 42 (35.3%) and six (5.0%) people achieved HBeAg and HBsAg loss at the end of follow-up (EOF; week 96). Baseline HBeAg level was independent predictor of VR (odds ratio [OR], 0.470; 95% confidence interval [CI], 0.294-0.751; P = 0.002), HBeAg loss (OR, 0.395; 95% CI, 0.243-0.643; P < 0.001), CR (OR, 0.392; 95% CI, 0.215-0.714; P = 0.002) at EOT and HBeAg loss at EOF (OR, 0.334; 95% CI, 0.203-0.559; P < 0.001); baseline HBsAg level itself was independent predictor of both HBsAg < 200 IU/mL at EOT (OR, 0.257; 95% CI, 0.103-0.642; P = 0.004) and HBsAg loss at EOF (OR, 0.232; 95% CI, 0.077-0.702; P = 0.010). Age was also independent predictors of HBsAg loss at EOF (OR, 0.775; 95% CI, 0.634-0.948; P = 0.013). Concerning genetic variation of VDR rs7975232/ ApaI, A allele was the genetic independent predictor of VR at EOT (OR, 1.824; 95% CI, 1.024-3.248; P = 0.041) and HBsAg loss at EOF (OR, 3.566; 95% CI, 1.057-12.029; P = 0.040). CONCLUSIONS: Genetic variation of VDR rs7975232/ ApaI is a pretreatment predictor of sustained HBsAg loss in HBeAg-positive CHB patients with Peg-IFN monotherapy.

[The effect of telbivudine on peripheral blood CD4+CD25+ regulatory T cells and its significance in patients with chronic hepatitis B].

OBJECTIVE: To investigate the effect of telbivudine on peripheral blood regulatory T cells and its significance in patients with chronic hepatitis B (CHB). METHODS: Thirty-six HbeAg positive chronic hepatitis B patients were recruited and received telbivudine treatment for 9 months. Before and during the 3, 6, 9 months of treatment, flow cytometry was used to detect the proportion of peripheral blood Tregs; real-time PCR was used to detect the levels of HBV DNA in the serum. Markers of hepatitis B virus infection were detected by ELISA assay and levels of alanine aminotransferase in the serum were measured. RESULTS: The proportion of peripheral blood Tregs in patients with CHB was significantly higher than that in healthy controls and decreased over 6 or 9 months of telbivudine treatment to a level comparable to that of the healthy controls. After 3 months of telbivudine treatment, the rate of undetectable HBV DNA in patients whose proportion of peripheral blood Tregs was decreased was higher than those whose Tregs had been reduced, but the difference was not statistically significant (P more than 0.05). Three, 6 or 9 months of telbivudine treatment resulted in HbeAg negativity in 4 (11.1%) patients, 7 (19.4%) patients or 9 (25.0%) patients respectively. In 7 (19.4%) patients who had seroconversion from HBeAg to anti-HBeAg, after 3 or 6 months of telbivudine treatment, their proportion of peripheral blood Tregs had decreased to a level comparable to that of the healthy controls. CONCLUSION: Telbivudine treatment reduces HBV replication and the proportion of peripheral blood Tregs. In addition, patients who have their proportion of peripheral blood Tregs decreased quickly at the early phase of telbivudine treatment are prone to have HBeAg to anti-HBeAg seroconversion.

[Effect of KLF6 and its splice variant KLF6V on proliferation and differentiation of human hepatocellular carcinoma HepG2 cells].

OBJECTIVE: To investigate the roles of Kruppel-like factor 6 (KLF6) and its splice variant KLF6V on suppressing growth and inducing differentiation of human hepatocellular carcinoma hepG2 cells. METHOD: KLF6V cDNA was amplificated by RT-PCR from human hepatocellular carcinoma (HCC) tissue and then sequenced. The recombinant vectors expressing KLF6 variant (KLF6V) were constructed using molecular clone technology based on established plasmid pcDNA3.1A(-)/wtKLF6. KLF6V or KLF6-transfected HepG2 cells were established after being screened with G418. Growth activity of HepG2/KLF6 or HepG2/KLF6V cells was detected by in vitro MTT assay. Expression of p21WAF1 or cyclin D1 protein was detected by Western blot, and expressions of AFP or ALB protein were measured by radioimmunoassay. RESULTS: A novel alternatively spliced transcript of the human KLF6 gene was found and its sequencing revealed that the variant form of KLF6 lacked 126nt and its encoded protein products had a deletion of 42 aa near the COOH-terminal amino acid in comparison with full-length KLF6. Although KLF6 alternative splicing was present in both normal and cancerous tissues, expression of the KLF6 splice variants seemed to be up-regulated in HCCs tissues. The isoform of KLF6 proteins antagonized the ability of wild-type KLF6 to up-regulate p21 expression or down-regulate cyclin D1 expression and suppress HepG2 cell proliferation. KLF6 gene increased albumin production and decreased alpha fetoprotein production of the cells. CONCLUSION: The isoform of KLF6 protein, present in HCC tissue, antagonizes the ability of wild-type KLF6 to suppress cell proliferation and induce cellular differentiation.

Role of interleukin-21 and interleukin-21 receptor polymorphisms in the treatment of HBeAg-positive chronic hepatitis B patients with peginterferon.

The aim of this study was to evaluate the relationship between interleukin-21 (IL-21) and interleukin-21 receptor (IL-21R) polymorphisms and the response to peginterferon alfa (PEG-IFN α) therapy in HBeAg-positive chronic hepatitis B (CHB) patients.A total of 143 HBeAg-positive CHB patients treated for 48 weeks with PEG-IFN α and followed up for 24 weeks post-treatment were retrospectively evaluated. Genotypes analysis was performed for IL-21 polymorphisms rs907715, rs2221903, and IL-21R polymorphisms rs3093301 and rs2285452. Serum IL-21 levels were measured by enzyme-linked immunosorbent assay.The end of virological response (EVR) rate was 46.9% (67/143) at the end of treatment, the sustained virological response (SVR) rate was 43.4% (62/143) and the complete response (CR) rate was 32.1% (46/143) at 24 weeks post-treatment. Patients who carried IL-21 rs 2221903 genotype AA had a rather higher rate of EVR (response rate: 52.4%, odds ratio [OR] 0.42, 95% confidence interval [CI]: 0.19-0.91, P = .021), SVR (response rate: 47.6%, OR 0.43, 95% CI: 0.19-0.95, P = .028), and CR (response rate: 38.1%, OR 0.31, 95% CI: 0.12-0.79, P = .014) when compared to those had AG genotype. Meanwhile, IL-21rs 2221903 genotype AA was also independently associated with markedly reduced HBsAg levels (>1og10 IU/mL) after 24 weeks treatment and low HBsAg levels (<100 IU/mL) at the end of treatment. IL-21 rs907715 AG/GG genotype was independently associated with SVR (OR: 2.92, 95% CI: 0.98-8.6, P = .039; OR: 3.23, 95% CI: 1.0-10.4, P = .039). Patients with IL-21 rs907715 AG/GG genotype had higher serum IL-21 levels than those with rs907715 AA genotype (P = .021).IL-21 rs2221903 and rs907715 polymorphisms were significantly associated with the treatment response to PEG-IFN α among Chinese HBeAg-positive CHB patients.

Inactivation of the tumor suppressor Krüppel-like factor 6 (KLF6) by mutation or decreased expression in hepatocellular carcinomas.

BACKGROUND AND AIM: The Krüppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity (LOH). However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas (HCCs). We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines. METHODS: We analyzed the exon-2 of KLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting. RESULTS: KLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression of KLF6 mRNA or protein was down-regulated in 8 (34.7%) or 9 (39.1%) of 23 HCC tissues. Wild-type KLF6 (wtKLF6) inhibited cellular proliferation and prolonged G1-S transition by inducing the expression of p21WAF1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant (mKLF6) had no effects. CONCLUSION: Our findings suggest that KLF6 may be involved in pathogenesis of HCC.

[Mutations of the tumor suppressor Kruppel-like factor 6 (KLF6) gene in hepatocellular carcinoma and its effect of growth suppression on human hepatocellular carcinoma cell line HepG2].

OBJECTIVE: To explore the mutated KLF6 gene in hepatocellular carcinoma (HCC) and to characterize its behavior in human hepatocellular carcinoma cell line HepG2. METHODS: We analyzed the DNA isolated from 23 hepatocellular carcinoma tissues and their adjacent nontumor tissues by polymerase chain reaction (PCR). Direct sequencing was used to establish the incidence of mutation in exon2 of the KLF6 gene. Loss of growth suppressive function of the HCC-derived KLF6 mutants was characterized by in vitro analyzing alteration of cell cycle and MTT assay. Expression of p21WAF1, a possible downstream gene of KLF6, was detected in human hepatocellular carcinoma cell line HepG2 transiently transfected with KLF6 genes. RESULTS: Mutations of KLF6 were found in 2 of the 23 (8.7%) hepatocellular carcinomas. The two mutations were located in the transactivation domain and one of them resulted in single amino acid substitution of TGG (W) by GGG (G) at codon 162. Unlike the wild-type KLF6, cancer-derived KLF6 mutants neither suppressed growth nor induced p21WAF1 following transfection into culture cells. CONCLUSIONS: Mutations of the KLF6 gene may play a role in the pathogenesis of HCC, but are not the dominating mechanism resulting in inactivation of KLF6 functions. KLF6 suppresses hepatocellular carcinoma cell proliferation partly through upregulating expression of the p21WAF1 gene.

[Application of PCR-based tRNAval Pol III-shRNA expression cassettes in screening of efficient siRNA for inhibiting hepatitis B virus].

OBJECTIVE: To screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs). METHODS: Based on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR. RESULT: Co-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration. CONCLUSION: The expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.

[Inhibition of HBs-GFP fusion gene expression by RNA interference].

OBJECTIVE: To develop an effective report gene system to test the effect of small interfering RNA (siRNA). METHODS: HBV S gene was fused with enhanced green fluorescent protein (EGFP) gene to form HBs-GFP and the plasmid containing HBs-GFP was constructed. A vector expressing small hairpin RNA (shRNA) pAVU6 + 4sh357 was also constructed. Two plasmids were co-transfected into HepG2 cells transiently. The fluorescence of HBs-GFP was detected by fluorescence-activated cell sorting (FACS). The mRNA expression in HepG2 cells was detected by conventional RT-PCR and real-time PCR. RESULTS: siRNA inhibited the expression of HBs-GFP 72 hours post transfection. The fluorescence of HBs-GFP in HepG2 cells treated with pAVU6+4sh357 was reduced by 55.4% compared with that of controls. The HBs-GFP expression in HepG2 cells treated with pAVU6+4sh357 was reduced by 76.3% and 90% as measured with conventional RT-PCR and real-time PCR, respectively. CONCLUSION: This investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells.

[Establish a screening system for selection of mRNA target sites for HBsAg to construct siRNA with shRNA].

OBJECTIVE: To find some effective short interfering RNA's sites targeting HBV surface gene sequence using shRNA expression vectors. METHODS: Four shRNA expression vectors targeting HBV surface gene sequence were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with HBs-EGFP fusion gene plasmid. The changes of HBs-EGFP image were detected by FACS and microscopy. The HBs-EGFP mRNA expression was evaluated by RT-PCR. RESULTS: Four shRNA expression vectors and HBs-EGFP fusion gene plasmid were successfully constructed. pAVU6 + 4sh579 vector inhibited the HBs-EGFP expression by 69.8% in AD293 and suppressed the HBs-EGFP mRNA expression by 74.6%. CONCLUSIONS: The results showed that the 579 site of HBV surface gene sequence was an effective target and pAVU6 + 4sh579 vector could suppress the HBs-EGFP expression in AD293 cells

Amino acid sequence analysis in patients with chronic HCV genotype 1b infection during pegylated interferon-α2a and ribavirin therapy.

BACKGROUND: Amino acid variations in several HCV genomic regions have been reported to be associated with response to interferon (IFN)-α plus ribavirin (RBV) combination therapy. However, the results remain controversial. In this study, we further investigated the amino acid variation of full-length HCV genome and its correlation to the response to pegylated interferon (PEG-IFN)-α2a and RBV combination therapy in patients with HCV genotype 1b (HCV-1b). METHODS: We retrospectively analysed 18 chronic HCV-1b patients (9 with rapid virological response and 9 non-response to therapy) treated with PEG-IFN-α2a plus RBV for 48 weeks. The nearly full-length HCV genome sequence was amplified by reverse transcription (RT)-PCR followed by cloning and sequencing. Genetic diversity differences between two groups were analysed including the number of amino acid variations in the HCV polyprotein and the mean pair-wise protein distance. RESULTS: No single amino acid variations were closely associated with treatment outcome. However, the number of amino acid mutations in the NS5B region especially in the thumb domain and in the NS5A-V3 region was associated with the response to PEG-IFN-α/RBV therapy (P=0.002 and P=0.029, respectively). The number of substitutions in the NS5B region was significantly correlated with the numbers of substitutions in the V3 region (r=0.568, P=0.027). CONCLUSIONS: Amino acid substitutions in the NS5B region especially in the thumb domain and the NS5A-V3 region may play a role in the response to combined PEG-IFN-α2a and RBV therapy in HCV-1b patients.

Synergistic effects of soluble PD-1 and IL-21 on antitumor immunity against H22 murine hepatocellular carcinoma.

Cancer immunotherapies are designed to elicit T-cell responses that inhibit tumor growth. Previous studies have demonstrated that interleukin 21 (IL-21) is a promising cytokine for cancer immunotherapy due to its ability to induce the immunity of T cells and natural killer cells, whereas blockade of the interaction of programmed death receptor-1 (PD-1) with its ligand (PD-L1) reduces peripheral tolerance. In the current study, we investigated IL-21 alone and in combination with soluble PD-1 (sPD-1) for the treatment of experimental H22 murine hepatocarcinoma. The naked plasmids pmIL-21 and/or psPD-1 were used for local gene transfer by injection. In these assays, sPD-1 combined with IL-21 was found to significantly inhibit the growth of the tumors in mice. Combined treatment with IL-21 and sPD-1 enhanced the antitumor immune response compared with that induced by IL-21 alone. Combined treatment was found to increase CTL cytotoxicity, increase the number of CTLs and NK cells in splenocytes, upregulate the cytokines IFN-γ and IL-2 and downregulate IL-10. Thus, immunotherapy with IL-21 in combination with sPD-1 was found to induce a more efficacious antitumor immune response, which may have potential clinical implications.