ABSTRACT: Changes of mRNA expression levels in HBV-infected human primary hepatocytes and those cells treated with bionanocapsules containing microRNA-93.
Project description:Human primary hepatocytes isolated from chimeric mice were infected with HBV for 7 days. The comprehensive changes of mRNA levels were determined by mRNA array. Also, microRNA93 was delivered into those cells using bionanocapsules to determine the effects of rescue expression of miR93 in HBV replicating cells, because we found that miR93 expression level was downregulated in HBV-infected hepatocytes. mRNA expression levels were compared in among control cells, HBV-infected cells, miR93 delivered cells, and HBV-infected cells with miR93 delivered, by 25K mRNA arrays.
Project description:Human primary hepatocytes isolated from chimeric mice were infected with HBV for 7 days. The comprehensive changes of mRNA levels were determined by mRNA array. Also, microRNA93 was delivered into those cells using bionanocapsules to determine the effects of rescue expression of miR93 in HBV replicating cells, because we found that miR93 expression level was downregulated in HBV-infected hepatocytes.
Project description:Human primary hepatocytes isolated from chimeric mice were infected with HBV for 7 days. The comprehensive changes of miRNA levels were determined by miRNA array. MicroRNA expression levels were compared in between control and HBV-replicating primary hepatocytes by 2K microRNA microarrays
Project description:Human primary hepatocytes isolated from chimeric mice were infected with HBV for 7 days. The comprehensive changes of miRNA levels were determined by miRNA array.
Project description:The in vitro HBV infection assay system was established in primary human hepatocytes (PXB cells) infected with HBV derived from the plasmid containing 1.3-mer HBV genome. Comprehensive and functional studies were performed by small interfering RNA knockdown and vector transfection experiments of the related genes and analyzed using microarray to reveal the host factors related to HBV regulation. Knockdown of STAT1 increased viral products in HBV-transfected HepG2 cells, but unexpectedly decreased in HBV-infected PXB cells. In RNA microarray analysis, STAT1 knockdown induced changes in intracellular molecules related to metabolism and protein synthesis in PXB cells, but not in HepG2 cells.
Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes expressing HBV to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes infected with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV). Samples were collected either 24h or 48h after infection. cDNA libraries were sequenced two times using the Illumina HiSeq or Illumina NextSeq platform to generate either 1x50bp or 1x75bp reads. Reads from each sequencing run were mapped using the STAR aligner, and output BAMs were merged into a single BAM for each sample. The merged BAM was further analyzed in R using the GenomicAlignments package to quantify number of reads per transcript and DESeq2 to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 24h and 48h post-infection. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of HBV-expressing and non-expressing primary rat hepatocytes were generated, in triplicate, 24h and 48h post-infection using Illumina HiSeq 2500 and NextSeq 500 instruments.
Project description:Several classes of capsid assembly modulators (CAMs) are currently being developed for chronic hepatitis B (CHB) cure. Both class A (CAM-A) and class E (CAM-E) CAMs disrupt nucleocapsid assembly and reduce extracellular hepatitis B virus (HBV) DNA. However, only CAM-As have been shown to reduce the number of HBV-infected cells in the animal models. However, there has been limited efficacy to date of CAM-A molecules achieving this secondary mechanism of HBV-infected cell clearance in CHB clinical trials. To investigate this disconnect, we performed comparative experiments with tool compounds from each class to further explore these unique features and antiviral activity of CAM-A across HBV-infected primary human hepatocytes (PHH), as well as in two different HBV mouse models (immunodeficient mice repopulated with human hepatocytes and AAV-HBV). Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced dose-dependent aggregation of HBV core protein (HBc) in the nucleus. Experiments with siRNA, resulted in identification of the ubiquitin-binding protein p62 as a factor negatively regulating the size of these aggregates. Furthermore, we found that only the CAM-A is able to induce HBc-positive cell death in vivo with the loss of HBV-infected cells positively correlated to the levels of intrahepatic HBc. Profiling of intrahepatic HBc levels across CHB patient liver biopsies demonstrated a significantly lower level of HBc per hepatocyte than either of the HBV mouse models. Taken together, these data demonstrate that CAMs of class A have a unique secondary mechanism that has potential to specifically affect viability of HBV-infected hepatocytes. At the same time, the clearance of infected hepatocytes may depend on the level of HBc expression thereby limiting the therapeutic potential for this class of molecules.
