Neelakshi Sarkar

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.

Influence of HIV-associated degree of immune suppression on molecular heterogeneity of hepatitis B virus among HIV co-infected patients

We have investigated the molecular diversity of Hepatitis B virus (HBV) among the HIV co-infected patients from eastern-India. HBsAg/HBV-DNA positive subjects (n=73) from 874 HIV-infected patients were analyzed by sequencing followed by genetic diversity quantification. HBV/genotype-D and HBV/sugenotype-D2 were predominant. HBV/D2 isolates from patients with low CD4 count manifested significantly lower non-synonymous substitutions (p<0.0001) and Shannon entropy (p=0.0006) in their surface and polymerase gene in comparison to those from moderately increased CD4 count. ART-induced immune-reconstitution therefore might raise non-synonymous immune/therapy escape substitutions among these HBV/D2 isolates. Decreased genetic diversity and increased viral load in the HBV/D2 isolates might facilitate the maintenance of their wild type characteristics in the low CD4 count, leading to its increased prevalence in this group. Interestingly, genetic diversity in HBV/A1, the next common subgenotype, was modified in the opposite manner. Together our results underscore the need for proper HBV molecular monitoring in HIV co-infection.

Hepatitis B Virus Infection, MicroRNAs and Liver Disease

Hepatitis B virus (HBV) attacks the liver and can cause both acute as well as chronic liver diseases which might lead to liver cirrhosis and hepatocellular carcinoma. Regardless of the availability of a vaccine and numerous treatment options, HBV is a major cause of morbidity and mortality across the world. Recently, microRNAs (miRNAs) have emerged as important modulators of gene function. Studies on the role of miRNA in the regulation of hepatitis B virus gene expression have been the focus of modern antiviral research. miRNAs can regulate viral replication and pathogenesis in a number of different ways, which includefacilitation, direct or indirect inhibition, activation of immune response, epigenetic modulation, etc. Nevertheless, these mechanisms can appropriately be used with a diagnosticand/or therapeutic approach. The present review is an attempt to classify specific miRNAs that are reported to be associated with various aspects of hepatitis B biology, in order to precisely present the participation of individual miRNAs in multiple aspects relating to HBV.

Characterization of Treatment-Naive HIV/HBV Co-Infected Patients Attending ART Clinic of a Tertiary Healthcare Centre in Eastern India

Objective The study was designed to assess the hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection scenario among the human immunodeficiency virus (HIV) infected patients attending a tertiary healthcare unit in eastern India. Additionally, clinical and virological characterization of these viruses, prior to antiretroviral therapy (ART) initiation was also done for better understanding of the disease profile. Methods Pool of ART-naive HIV/HBV co-infected and HIV mono-infected patients, participating in two different studies, were included in this study. HBV DNA was detected by nested-PCR amplification followed by HBV genotype determination and HBV reverse transcriptase (RT) region amplification and direct sequencing for detecting drug resistance. Results The prevalence of HBsAg (11.3%) was higher compared to anti-HCV (1.9%) among the HIV infected ART-naive patients. Moreover, majority of the HBeAg positive HIV/HBV co-infected patients (87.7%) had HBV DNA ≥20,000 IU/ml with median HBV DNA significantly higher than that of HBeAg negative subjects (5.7 log10 IU/ml vs. 4.2 log10 IU/ml; p<0.0001). Multivariate analysis also showed that HBeAg-positive status was independently associated with higher HBV DNA level (p = <0.001). Notably, 60.9% of the HBeAg negative co-infected subjects had HBV DNA ≥2,000 IU/ml of which 37.0% had HBV DNA ≥20,000 IU/ml. Genotype HBV/D (68.2%) was the predominant genotype followed by HBV/A (24.3%) and HBV/C (7.5%). Anti-HBV drug resistant mutations were detected in two (3.8%) of the ART-naive patients. Conclusion The prevalence of HIV/HBV co-infection was relatively higher in our study subjects. HBeAg testing might provide clue for early treatment initiation. Furthermore, HBeAg negative patients are also associated with high HBV DNA levels and therefore require appropriate medical attention. Pre-treatment screening for anti-HBV drug resistant mutations is not necessary before ART initiation.

Molecular Characterization of HBV Strains Circulating among the Treatment-Naive HIV/HBV Co-Infected Patients of Eastern India

Previously we reported that the exposure to hepatitis B virus (HBV) infection serves as a major threat among the treatment naive HIV infected population of eastern India. Hence, molecular characterization of these strains is of utmost importance in order to identify clinically significant HBV mutations. A total of 85 treatment naive HIV/HBV co-infected participants were included of whom the complete basal core promoter/precore region, the core and the whole envelope gene could be successfully sequenced for 59, 57 and 39 isolates respectively. Following phylogenetic analysis, it was found that HBV/D was the predominant genotype with HBV/D2 (38.5%) being the most prevalent subgenotype followed by HBV/A1. The major mutations affecting HBeAg expression includes the A1762T/G1764A (13.6%), G1896A (22%) and G1862T mutation (33.9%) which was predominantly associated with HBV/A1. Moreover, the prevalence of G1896A was considerably high among the HBeAg negative HIV/HBV co-infected subjects compared to HBV mono-infection. The main amino acid substitutions within the MHC class II restricted T-cell epitope of HBcAg includes the T12S (15.8%) and T67N (12.3%) mutation and the V27I (10.5%) mutation in the MHC class I restricted T-cell epitope. PreS1/S2 deletion was detected in 3 isolates with all harboring the BCP double mutation. Furthermore, the frequently occurring mutations in the major hydrophilic loop of the S gene include the T125M, A128V and M133I/L. Therefore, this study is the first from India to report useful information on the molecular heterogeneity of the HBV strains circulating among the treatment naive HIV/HBV co-infected population and is thus clinically relevant.

Virological Characteristics of Acute Hepatitis B in Eastern India: Critical Differences with Chronic Infection.

Hepatitis B Virus (HBV) manifests high genetic variability and is classifiable into ten genotypes (A-J). HBV infection can lead to variable clinical outcomes, ranging from self-limiting acute hepatitis to active chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study characterizes HBV strains circulating among patients with acute (AHB) and chronic HBV infection (CHB). Among a total of 653 HBsAg positive cases, 40 manifested acute infection. After sequencing the surface(S), basal core promoter/pre-core(BCP/PC) and the X gene regions, phylogenetic tree was constructed using MEGA4 by neighbor-joining method. Statistical robustness was established with bootstrap analysis. Nucleotide diversity was determined by Shannon entropy per site using the Entropy program of the Los Alamos National Laboratories. Analyses of acute patients revealed that HBV/D2 is the major circulating sub-genotype and commonly associated with sexual promiscuity and the age group between15-30 years. Comparison of AHB and CHB patients revealed that HBeAg positivity, ALT levels and genotype D were significantly high in AHB, whereas CHB patients were predominantly male, had a high viral load, and were commonly associated with genotype C. The frequencies of mutations in the S, BCP/PC, and X gene were low in AHB as compared to CHB. Drug resistant mutations were not detectable in the polymerase gene of AHB. Average nucleotide diversity in AHB was considerably low as compared to CHB. Further, the highest average ΔH (average difference in entropy between chronic and acute infection) was observed in the BCP/PC region implying that this region was most vulnerable to mutations upon HBV persistence, especially in case of genotype C. Additionally, among all substitutions, the A1762T and G1764A BCP mutations were the strongest indicators of chronicity. In conclusion, the study exhibits a general portrait of HBV strains circulating among acute hepatitis B patients in Eastern India and their intricate differences with chronic patients which should be useful from the clinical point of view.

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.

Occult hepatitis B virus infection in HIV positive patients at a tertiary healthcare unit in eastern India

Occult HBV infection (OBI), defined by the presence of HBV DNA in absence of hepatitis B surface antigen (HBsAg), is a significant concern in the HIV-infected population. Of 441 HIV+/HBsAg- patients analyzed, the overall prevalence of OBI was 6.3% (28/441). OBI was identified in 21 anti-HBc positives (17.8%), as well as among those who lacked any HBV-specific serological markers (2.2%). Comparison with HIV/HBV co-infection revealed that the levels of CD4, ALT, and HBV DNA were significantly lower during occult infection. Discrete differences were also observed with respect to quasispecies divergence. Additionally, subgenotype D1 was most frequent in occult infection, while D2 was widespread during chronic infection. The majority (~90%) of occult D1 sequences had the sQ129R mutation in the surface gene. This study highlights several distinct features of OBI in India and underscores the need for additional HBV DNA screening in HIV-positive individuals.

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.