Effects if hepatitis C virus RNA on gene expression profile of infected human hepatocytes

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Effects if hepatitis C virus RNA on gene expression profile of infected human hepatocytes

Introduction

Many recent research and studies shows that hepatitis C virus (HCV) can interfere with, or change the expression of various genes including those of infected humans’ hepatocytes. To understand this research studies better, analyzing the direct effects of interferon or hepatitis C virus (HCV) under immunodeficient condition, or provision using cDNA microarray analysis of human hepatocyte chimeric mice is undertaken. All over the world chronic hepatitis C is one of the marked worldwide health threats, affecting over one hundred and fifty million globally.

The attempt to eradicate the virus and to prevent the development of advanced liver diseases, for instance chronic hepatics; interferon is administered to the chronic patients. The virus in the recent studies has been found to affect innate immunity or lipid metabolism in people. The probes in this paper include, transcriptional profiling, comparative genome hybridization, alternative splicing, transcriptome annotation, small microRNA profiling, methylation pattern, chip chip probing, genotyping, intronic transcription, among others. These probing types were chosen based on previous studies on the same study as this one, where these probing methods were the most preferred ones. The probes have unique exact match target that also made them ideal for this design (Boyer, 2001).

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Epigenomic microarrays

Only a minor fraction of hepatitis C virus is occupied by genes; however,

histone and non histone chromosomal proteins and methylated DNA bases are distributed over both genic and intergenic regions. Upon marking, the microarray platform can be used to obtain the profiling patterns of these widespread epigenomic features, such as DNA methylation, DNA replication, DNA binding, and chromatin-associated proteins and histone modifications. Alternatively to the already-mapped sites, microarray-based strategies are able to identify novel DNA binding sites or novel DNA methylation regions by probing upstream and downstream regions of genes. Certainly, epigenomic microarrays will become a standard research tool for understanding chromatin structure and gene expression during development. Similar to gene-oriented arrays, epigenomic microarrays are easy-to-handle tools in comparison to tiling arrays and permit that many different experiments be performed at a low cost and lower labour analysis.

However, for identification of the complete set of epigenomic features of an organism, a tiling-array platform is the best tool since it covers long contiguous genomic regions (Branch, 2001).

Tiling Arrays

With the completion of sequencing of many genomes, attention has shifted to determining the complete set of transcribed sequences and regulatory elements. This recent trend in genomics has involved the development of tiling arrays: microarrays that represent a complete non-repetitive tile path over a locus, chromosome or whole-genome, irrespective of any genes that may be annotated in that region. Potential uses for such unbiased representation of gDNA include empirical annotation of the transcriptome, chromatin immunoprecipitation-chip studies, characterization of the methylation state of CpG islands, analysis of alternative splicing, and CGH.

Numerous options exist for tiling genomic sequences with oligonucleotides or PCR products, leading to microarray designs of different sequence resolutions and feature densities. Oligonucleotide arrays comprise 25–70 bp probes, which are synthesized directly on the slides or prepared in solution and then deposited. The second type of tiling array is constructed using PCR products typically of ~1 kb in length, or BAC arrays – typically at 1 Mb resolution. One caveat of PCR-based tiling arrays is that their construction is labour intensive and therefore they are not readily scalable to the study of large (Carr, 2010).

Small microRNA Profiling

This involves grouping together of the same RNA and checking if the hepatitis C virus is occupied by genes matches the profiled microRNA. This probing method is chosen because it chooses it singles out the infected genes, making diagnosis of the infection easy (Springer, 2010).

Comparative genome hybridization

This method allows for detection of chromosomal copy number changes on the hepatitis C virus, it has a high resolution scale. The high resolution scale is what makes it is used in this experiment (Carr, 2010).

Methylation Pattern

DNA methylation in the expression profile of infected human hepatocytes in person infected with hepatitis C virus is an epigenetic mark crucial in regulation of gene expression. It is used in this experiment since it is important for silencing of repetitive elements such as transposons and retroviruses, and for epigenetic. It works on basis of microRNAs detection by microarrays (Deonier et al, 2005).

Chip chip

Chromatin immunoprecipitation (ChIP) coupled to hybridization onto DNA microarrays (ChIP-chip) is becoming a popular approach to investigate interactions between proteins and DNA that occur infected genes by the hepatitis C virus. In ChIP-chip experiments, cross-linked chromatin–protein complexes are extracted from a cell or tissue of interest and the DNA sheared, typically by sonication, down to relatively short. DNA fragments cross-linked to the protein of patients (Falk et al, 2001).

Discussion

Hepatitis C virus spreads faster since the virus multiplies at a faster rate compared to other disease causing compounds. The fast spreading enables the viruses to develop before the body comes up with defense. Hepatitis treatment is difficult since virus spreads it and they replicate faster making the viruses spread fast compared to the white blood cells that fight them in the body. The HCV mostly affect the RNA therefore, they are able duplicate and move genetically to offspring’s of the next generation. The new culture performed encompassed the non-cancerous and were renewable culture system (Schinazi et al, 2003).

In primary hepatocyte cultures experimented, the duplication of the HCV is not identified and it was found through the polymerization of the chain reaction (qRT-PCR). This was done on the HCV RNA, though it was not appropriate for perceiving such extraordinary virus. To curb this they used the HCVcc that is a very much susceptible and is produced Gaussia luciferase( Gluc), JclFLAG2(p7-nsGluc2A). The mixture of the culture is then to get rid of Gluc carryover and luciferase and the observed as a display of viral replication. It was identified as various culture systems namely Matrigel overlay, collagen cell sandwich and other erratically distributed co cultures randomly.

The above named components could not maintain HCV repetition due to the decline of the precise liver phenotypes. Other results proved that MPCCs in various form could not support HCV repetition for a period of two weeks. The cure of the HCV using the NS5B polymerase and the NS3-4 protease or alpha (IFN) lead to a reduction of the effect of the luciferase. The effects of the luciferase took a longer period due to the faster repetition of the viruses. HCV affects the mRNA and this is evident in the liver of a HCV- infected rat whereby there was higher numbers of the MRP4 and OATP2B1 and lower numbers of OCT1. There was a similarity between the actions of human cytochrome P45O enzymes and the non-infected CYP1A2 (Thomas et al, 2008).

The change of the mRNA activity may not be adequate to influence its enzymatic activity whereas the transcriptional effects of of HCV infection on the expression of pharmacokinetics-related genes would also be observed at the transcriptional level. HCV affects the genes through direct border with the viruses and the innate antiviral response or direct bordering by adaptive HCV-specific immune response. It may also be due to the liver diseases associated with chronic infection and oxidative stress.

The induction of the microRNA by hepatic conditions led to the transition from non-persiveness to permissiveness. The genes responsible for the cell signaling pathways and extra cellular components were found in plenty after addition of used in the culture. Some receptors such as the cluster of differentiation 81(CD81), SR-BI, claudin-1 and occludin stay mostly unchanged. The epidermal growth factor receptor (EGFR), ephrin receptor A2 (EphA2) and two other receptors tyrosine kinases (RTKs) identified in siRNA library that display for HCV entry factors within ten days (Thomas et al, 2008).

Appearance of the cellular co factors of HCV remained unchanged since the RNA data from the microarray and conventional RT-PCR. shRNA was identified to block HCV infection in human hepatoma cell by tapping down the expression of the CyPA. CyPA is important in the HCV life cycle and thus the shRNA blocks its functions reducing its influence on the HCV virus (Thomas et al, 2008).

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(Deonier et al, 2005)

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