Manikankana Bandopadhyay

Tumor suppressor micro RNA miR-145 and onco micro RNAs miR-21 and miR-222 expressions are differentially modulated by Hepatitis B virus X protein in malignant hepatocytes

BackgroundHepatitis B Virus (HBV) X protein (HBx) is known to be involved in the initiation and progression of hepatocellular carcinoma (HCC) through modulation of host gene response. Alterations in miRNA expressions are frequently noted in HCC. This study is aimed to examine the role of HBx protein in the modulation of oncogenic miRNA-21, miRNA-222 and tumor suppressor miRNA-145 in malignant hepatocytes.MethodsExpressions of miRNA-21, miRNA-222 and miRNA-145 were measured in HepG2 cells transfected with HBx-plasmid (genotype D) and with full length HBV genome (genotype D) and also in stably HBV producing HepG2.2.15 cells using real time PCR. Their target mRNAs and proteins - PTEN, p27 and MAP3K - were analyzed by real time PCR and western blot respectively. miRNA expressions were measured after HBx/D mRNA specific siRNA treatment. The expressions of these miRNAs were analyzed in liver cirrhosis and HCC patients also.ResultsThe study revealed a down-regulation of miRNA-21 and miRNA-222 expressions in HBx transfected HepG2 cells, pUC-HBV 1.3 plasmid transfected HepG2 cells as well as in HepG2.2.15 cells. Down regulation of miRNA-21 and miRNA-222 expression was observed in patient serum samples. Down regulation of miRNA-145 expression was observed in HepG2 cells transiently transfected with HBx and pUC-HBV1.3 plasmid as well as in patient samples but the expression of miRNA-145 was increased in HepG2.2.15 cells. Target mRNA and protein expressions were modulated in HepG2 cells and in HepG2.2.15 cell line consistent with the modulation of miRNA expressions.ConclusionThus, HBx protein differentially modulated the expression of miRNAs. The study throws light into possible way by which HBx protein acts through microRNA and thereby regulates host functioning. It might suggest new therapeutic strategies against hepatic cancer.

Expression of microRNA-155 correlates positively with the expression of Toll-like receptor 7 and modulates hepatitis B virus via C/EBP-β in hepatocytes

Effective recognition of viral infection and successive activation of antiviral innate immune responses are vital for host antiviral defence, which largely depends on multiple regulators, including Toll‐like receptors (TLRs) and microRNAs. Several early reports suggest that specific TLR‐mediated immune responses can control hepatitis B virus (HBV) replication and express differentially with disease outcome. Considering the versatile function of miR‐155 in the TLR‐mediated innate immune response, we aimed to study the association between miR‐155 and TLRs and their subsequent impact on HBV replication using both a HBV‐replicating stable cell line (HepG2.2.15) and HBV‐infected liver biopsy and serum samples. Our results showed that miR‐155 was suppressed during HBV infection and a subsequent positive correlation of miR‐155 with TLR7 activation was noted. Further, ectopic expression of miR‐155 in vitro reduced HBV load as evidenced from reduced viral DNA, mRNA and subsequently reduced level of secreted viral antigens (HBsAg and HBeAg). Our results further suggested that CCAAT/enhancer‐binding protein‐β (C/EBP‐β), a positive regulator of HBV transcription, was inhibited by miR‐155. Taken together, our study established a correlation between miR‐155 and TLR7 during HBV infection and also demonstrated in vitro that increased miR‐155 level could help to reduce HBV viral load by targeting C/EBP‐β.

Hepatitis B virus X protein mediated suppression of miRNA-122 expression enhances hepatoblastoma cell proliferation through cyclin G1-p53 axis

BackgroundHepatitis B virus (HBV) X protein (HBx) reported to be associated with pathogenesis of hepatocellular carcinoma (HCC) and miR-122 expression is down regulated in HCC. Previous studies reported miR-122 targets cyclin G1 (CCNG1) expression and this in turn abolishes p53-mediated inhibition of HBV replication. Here we investigated the involvement of HBx protein in the modulation of miR-122 expression in hepatoblastoma cells.MethodsExpression of miR-122 was measured in HepG2 cells transfected with HBx plasmid (HBx-HepG2), full length HBV genome (HBV-HepG2) and in constitutively HBV synthesizing HepG2.2.15 cells. CCNG1 mRNA (a direct target of miR-122) and protein expressions were also measured in both HBx-HepG2, HBV-HepG2 cells and in HepG2.2.15 cells. miR-122 expressions were analyzed in HBx-HepG2, HBV-HepG2 and in HepG2.2.15 cells after treatment with HBx mRNA specific siRNA. Expressions of p53 mRNA and protein which is negatively regulated by CCNG1 were analyzed in HBx transfected HepG2 cells; X silenced HBx-HepG2 cells and X silenced HepG2.2.15 cells. HBx induced cell proliferation in HepG2 cells was measured by cell proliferation assay. Flow cytometry was used to evaluate changes in cell cycle distribution. Expression of cell cycle markers were measured by real time PCR.ResultsExpression of miR-122 was down regulated in HBx-HepG2, HBV-HepG2 and also in HepG2.2.15 cell line compared to control HepG2 cells. CCNG1 expression was found to be up regulated in HBx-HepG2, HBV-HepG2 cells and in HepG2.2.15 cells. Following siRNA mediated silencing of HBx expression; increased miR-122 levels were documented in HBx-HepG2, HBV-HepG2 and in HepG2.2.15 cells. HBx silencing in HBx-HepG2 and HepG2.2.15 cells also resulted in increased p53 expression. FACS analysis and assessment of expressions of cell cycle markers revealed HBx induced a release from G1/S arrest in HepG2 cells. Further, cell proliferation assay showed HBx promoted proliferation of HepG2 cell.ConclusionOur study revealed that HBx induced down regulation of miR-122 expression that consequently increased CCNG1 expression. This subsequently caused cell proliferation and release from G1/S arrest in malignant hepatocytes. The study provides the potential to utilize the HBx-miR-122 interaction as a therapeutic target to limit the development of HBV related HCC.

Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line

BackgroundToll like receptors (TLRs) play an important role in innate immunity and various studies suggest that TLRs play a crucial role in pathogenesis of hepatitis B virus (HBV) infection. The present study aims in looking into the status of crucial host and viral gene expression on inciting TLR7.MethodsThe transcription of TLR7 pathway signaling molecules and HBV DNA viral load were quantified by Real Time-PCR after stimulation of TLR7 with its imiquimod based ligand, R837. Cell cycle analysis was performed using flow-cytometry. Expression of TLR7 and chief cell cycle regulator governing G1/S transition, p53 was also seen in liver biopsysss samples of CHB patients. HBV induced alteration in histone modifications in HepG2 cells and its restoration on TLR7 activation was determined using western blot.ResultsThe TLR7 expression remains downregulated in HepG2.2.15 cells and in liver biopsy samples from CHB patients. Interestingly HBV DNA viral load showed an inverse relationship with the TLR7 expression in the biopsy samples. We also evaluated the anti-viral activity of R837, an agonist of TLR7. It was observed that there was a suppression of HBV replication and viral protein production upon TLR7 stimulation. R837 triggers the anti-viral action probably through the Jun N-terminal Kinase (JNK) pathway. We also observed a downregulation of histone H3K9Me3 repression mark upon R837 treatment in HBV replicating HepG2.2.15 cells, mimicking that of un-infected HepG2 cells. Additionally, the G1/S cell cycle arrest introduced by HBV in HepG2.2.15 cells was released upon ligand treatment.ConclusionThe study thus holds a close insight into the changes in hepatocyte micro-environment on TLR7 stimulation in HBV infection.

Anti-viral role of toll like receptor 4 in hepatitis B virus infection: An in vitro study

AIM Toll like receptors plays a significant anti-viral role in different infections. The aim of this study was to look into the role of toll like receptor 4 (TLR4) in hepatitis B virus (HBV) infection. METHODS Real time PCR was used to analyze the transcription of TLR4 signaling molecules, cell cycle regulators and HBV DNA viral load after triggering the HepG2.2.15 cells with TLR4 specific ligand. Nuclear factor (NF)-κB translocation on TLR4 activation was analyzed using microscopic techniques. Protein and cell cycle analysis was done using Western Blot and FACS respectively. RESULTS The present study shows that TLR4 activation represses HBV infection. As a result of HBV suppression, there are several changes in host factors which include partial release in G1/S cell cycle arrest and changes in host epigenetic marks. Finally, it was observed that anti-viral action of TLR4 takes place through the NF-κB pathway. CONCLUSION The study shows that TLR4 activation in HBV infection brings about changes in hepatocyte microenvironment and can be used for developing a promising therapeutic target in future.

49 HEPATITIS B VIRUS X PROTEIN INDUCED MODULATION IN THE EXPRESSION OF MIR-222 IN MALIGNANT HEPATOCYTES IN VITRO

S 20TH ANNUAL CONFERENCE OF INASL, MARCH 2–4, 2012 S28

A Study on HBV Infection in HBV-HIV Co-infected Patients from Eastern India

© 2011, INASL 3 negative samples. For HBV DNA positive cases, HBeAg status was checked and direct sequencing of surface gene, BCP and PC region were done. Surface gene sequences were used to confirm the HBV genotypes/subgenotypes by phylogenetic analysis. The viral load was measured by real time PCR. Results: After initial screening, 204 samples were found to be anti-HBc positive but HBsAg negative. From these 204 samples 31 (15.2%) were found to be positive for HBV DNA. HBV genotype A was present in 9.7% and rest of the samples were HBV/D (90.3). Three subgenotypes of HBV/D was identified, viz D2 (35.7%), D3 (50.0%) and D5 (14.3). In the BCP region most common mutations were C1752, C1753 and triple mutation C1753/T1762/A1764. In 48.4% cases the presence of C1773 was noticed. Notably all the samples were found to be HBeAg negative. No mutation was found in the PC region. Conclusion: Interestingly higher rate of variability in the BCP region, especially C1752, C1753/T1762/A1764 triple mutations and C1773 was noticed. These carriers carrying the mutant strains should be followed up at regular intervals to assess the clinical progression. Conflict of Interest: None A Study on HBV Infection in HBV-HIV Co-infected Patients from Eastern India A Pal*, M Bandopadhyay*, A Biswas*, R Panigrahi*, SK Guha**, S Chakrabarti†, R Chakravarty* *ICMR Virus Unit, Kolkata **Department of Medicine, School of Tropical Medicine, Kolkata †National Institute of Cholera and Enteric Diseases, Kolkata Background: Hepatitis B virus (HBV) and HIV co-infection is frequent due to shared routes of transmission. With improved control of HIV disease with HAART, liver disease has emerged as one of the leading causes of deaths in HIV patients. It has been reported that co-infection with HIV complicates the natural history, diagnosis and management of HBV infection. In India, the study regarding HBV-HIV co-infection is sparse although the prevalence of both viruses is considerable. Objective: The study is aimed to characterize HBV infection in hepatitis B surface antigen (HBsAg) positive cases of HBV-HIV co-infected patients. Methods: Total 48 HIV patients who were registered in our unit for HBV screening were included. Serological markers of HBV and HIV were tested using enzyme linked immunoassays. Detection of HBV DNA was done by nested PCR, followed by genotyping using RFLP. Result: Thirty-two HIV patients were detected positive for HBsAg and HBV DNA resulting in 66.67% of selected HIV patients to be co-infected with HBV. 75% of 32 co-infected patients were also positive for HBeAg. Sexual transmission (47.62%) was found to be more associated with HBV transmission than blood transfusion and surgical operation (9.52% in both cases). When HBV genotype distribution was correlated with CD4 cell count, three genotypes [HBV/A (21.34%), HBV/C (7.14%) and HBV/D (64.29%)] were found to be related with CD4 cell count 6 months included. Patients with cirrhosis, hepatocellular carcinoma, antiviral treatment, HIV/HCV coinfection, excluded. Detailed clinical examination, LFT, USG abdomen, AFP, HBsAg ELISA titers, HBeAg and HBV DNA PCR (Roche Amplicor) assays done. OGD done when indicated. Anti-HBe antibody assessed in HBeAg negative patients. Pearson’s correlation coefficient (r) used for correlation between variables, analyzed using SPSS. 03_JCEH-Abstract.indd 3 3/18/2011 11:13:03 AM

Variability of BCP and Precore Region Among the Occult HBV Infected Voluntary Blood Donors from Eastern India

© 2011, INASL 2 PBMCs/well were stimulated with ORF-2 peptide (452–617 a.a) and subjected to MTT assay. Proliferation index (PI) at 570 nm was calculated. All the samples were subjected for anti-HEV IgM, IgG serology and HEV RNA detection by RT-PCR. Results: Patients with hepatitis were diagnosed as acute hepatitis E based on anti-HEV IgM/RNA positivity. PBMCs of acute hepatitis patients showed reactivity to the ORF-2 peptide in comparison to control subjects in terms of blast cell transformation. The proliferation indices of patient and control groups were 3.0398 ± 0.666 and 1.21 ± 0.077, respectively. Thus significant antigenic reactivity (p = 0.0126) was observed in hepatitis E patients. Anti-HEV IgM was positive in 15 (93.75%), IgG in 10 (62.5%) and RNA in 7 (43.75%) patients. None of the parameters were positive in control group. There was no significant difference in PI values between patient groups with and without HEV RNA positivity (2.99 ± 0.664, 3.077 ± 0.705; p = 0.359). Conclusion: HEV specific ORF 2 peptide (452–617 a.a) effectively stimulates the PBMCs in the patient group as compared to healthy controls. Thus, the tested peptide was found to be antigenic and specific for HEV and can be used in understanding the pathogenesis of HEV infection in vitro with regards to gene modulation and various cytokine profiles. Conflict of Interest: None Gene Expression Analysis in Peripheral Blood Mononuclear Cells in Patients with Acute Hepatitis E A Naik*, A Goel*, D Goel*, A Pandey**, S Rastogi†, R Aggarwal* *Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow **Department of Obstetrics and Gynecology, King George’s Medical College, Lucknow †Command Hospital, Lucknow Background: Hepatitis E is the commonest cause of acute viral hepatitis in India and several developing countries. Pathogenesis of liver injury and immune events during this disease are not well understood. To better define immune events during hepatitis E, we undertook a gene expression study of peripheral blood mononuclear cells in patients with this disease. Methods: PBMCs were obtained from 12 patients with acute hepatitis E (typical clinical and biochemical findings, and positive IgM anti-HEV [Genelabs, Singapore]), 4 patients with acute hepatitis B (HBsAg positive, IgM anti-HBc positive, IgM anti-HEV negative) and 10 healthy controls, using Ficoll-Hypaque density gradient centrifugation. Total RNA was isolated from PBMCs using Qiagen RNeasy Protect Mini Kit, and cDNA synthesis and microarray analysis were done using Illumina HumanWG-6 v3.0 Expression BeadChips. Genes showing differential expression in hepatitis E, but not in hepatitis B, were identified and downstream pathway analysis was done. Results: PBMCs from patients with hepatitis E showed differential expression of 170 genes (upregulated 140, downregulated 30). PBMCs from patients with hepatitis B showed differential expression of 80 genes (up 62, down 18). Of these, 24 differentially-expressed genes were common. Genes differentially expressed in PBMCs from patients with hepatitis E but not in hepatitis B belonged to immune pathways that included cell-mediated immune response, immune cell trafficking and antigen presentation. The genes that were overexpressed in PBMCs from patients included CDCA5, CCNA2, CCNB2, PCNA, IGLL1 and TNFRSF17; most of these genes are involved in cell division and cellular function. In comparison, most of the genes that were underexpressed in PBMCs from hepatitis E (CCL20, TNF, IL1RN, CCL4L1, CCL3L3, CCL3, CCL3L1, IL1RN and IL1B) belonged to chemokine and pro-inflammatory cytokine families. Conclusion: Downregulation of chemokines and pro-inflammatory cytokines in PBMCs from patients with hepatitis E may contribute to disease pathogenesis by allowing enhanced viral replication. Conflict of Interest: None Variability of BCP and Precore Region Among the Occult HBV Infected Voluntary Blood Donors from Eastern India A Biswas*, R Panigrahi*, A Pal*, M Bandopadhyay*, BK De**, S Chakrabarti†, R Chakravarty* *ICMR Virus Unit, Kolkata **Department of Medicine, Calcutta Medical College, Kolkata †National Institute of Cholera and Enteric Diseases, Kolkata Background: In India prevalence of occult hepatitis B virus (HBV) infection in the general population is widespread. Data about the variability of the basal core promoter region (BCP) and precore (PC) region of the occult HBV strains is lacking. Objective: Mutations in the BCP and PC regions are associated with persistent/intermittent HBV replication. We evaluated the variability BCP and PC region of HBV in asymptomatic carriers and aimed to characterize the variability with different viral strains. Methods: From voluntary blood donation camps initially 1400 blood samples were collected. The anti-HBc and HBsAg status of the samples were confirmed by ELISA. PCR amplification to test the presence of HBV DNA was done from anti-HBc positive but HBsAg 03_JCEH-Abstract.indd 2 3/18/2011 11:13:03 AM

Characterization of S Gene Region Among HBV-infected Patients from Orissa

© 2011, INASL 12 Characterization of S Gene Region Among HBV-infected Patients from Orissa R Panigrahi*, A Biswas*, MK Panigrahi**, A Pal*, M Bandopadhyay*, SP Singh**, HS Das**, S Chakrabarti†, R Chakravarty* *ICMR Virus Unit, Kolkata, ID and BG Hospital Campus, Kolkata **SCB Medical College, Cuttack, Orissa †National Institute of Cholera and Enteric Diseases, Kolkata, India Background: Genetic mutation might account for the presence of hepatitis B virus (HBV) DNA among liver disease individuals. The aim of this work was to describe the clinical characteristics associated with the HBV genotypes circulating in south-eastern India. Materials: A total of 36 HBsAg patients were included in the study. Surface gene regions were amplified by PCR for detection of HBV infection and surface gene was analyzed after direct sequencing. Analysis of MHR region was analyzed by using the BLOSUM scores. Results: HBV genotypes observed in HBV-infected patients were as follows: 8 patients with genotype A (22.2%), 1 with C (2.7%), 27 with D (75.0%). Male patients predominated in this study (91.6%). Among the 18 patients infected with sub-genotype D5, a considerably higher prevalence was found among LC patients (55.5%), CHB patients (27.7%), and acute patients (16.6%). No prevalence of HBV/D2 was found in the present study. The distribution of MHR mutations among the clinical groups was found in acute patients (0%), CHB patients (10%), and LC patients (17.6%). Conclusion: HBV sub-genotypes D5 were found in a significant proportion in our study population as compared with other parts of India. Interestingly, HBV D5 was found to be more frequently associated with liver cirrhosis than other clinical groups. Mutations in the immunodominant domains of S gene were found to increase with increasing disease severity. Thus our study will add to the knowledge of HBV genotype distribution and characterization in India. Conflict of Interest: None 03_JCEH-Abstract.indd 12 3/18/2011 11:13:03 AM