| Autonomous parvoviruses are nonenveloped, linear-stranded, lytic DNA viruses which are cytotoxic to neoplastic cells while innocuous to their non-transformed counterparts, thus become a candidate of choice as vehicles in gene therapy. It is therefore of special importance to uncover the mechanism underlying parvovirus-host cell interaction. Previous studies have identified dozens of cellular factors which are necessary and helpful in viral life cycle and the some roles of viral proteins on the host cell have also been determined. However, the global impact of parvovirus infection on the host cell has not been investigated by far. Liver cancer is one of the most common cancer in China and southern Asia, the relationship between several hepatoma cell lines and parvovirus H-l have been investigated in the previous works, among them human hepatocellular carcinoma cell line QGY-7703 is the most well-studied one. In order to gain insight into the global cellular response at the transcriptional level and the possible cellular targets after parvovirus infection, QGY-7703-H-l was used as a model system and DNA microarray technology which simultaneously measure the transcript abundance of thousands of genes was employed to identify the target genes and pathways which underlie the mechanism of parvovirus-host interaction.The replication of parvovirus is strongly dependent on cellular factors associated with proliferation and differentiation, especially those related to the S-phase of the cell cycle. Conversely, the multiplication of virus can induce cell cycle arrest at S/G2 phase. With the aim of enhancing the fraction of cells expressing the viral genome as well as reducing the differences between samples under comparison due to virus interference with the cell cycle, synchronized cells were used to perform the experiments. Several approaches including serum starvation, isoleucine depletion, drug treatment and isoleucine plus drug treatment were tried to find the best method and optimal condition for synchronizing cells. Mimosine was found suitable to yield a highly synchronized QGY-7703 cell population. Cells were arrested at late Gl phase after drug treatment and proceed synchronously during at least one cycle. The cells were infected in the presence of this drug. The morality of cells and the replication of virus in synchronized cells after infection were monitored. The results show that after release from the cell cycle arrest cells NS1 protein and its mRNA increase over time,cell morality is low before 12h postrelease but increase over time after this time point. Two time points (6hrs and 12hrs postrelease) at which cells are synchronously in the same cell cycle stage, the NS1 expression is high and the morality is still low were selected for further study.The gene expression profiles at these time points were measured respectively using oligonucleotide microarray (Genechip Human Genome U133A, Affymetrix) that represent 22,000 human genes. In order to improve the reliability of the experiments, the quality of the samples, mainly the integrity of RNA at various steps in the experiment was monitored, the results demonstrated high quality of the samples. The experiment was performed in duplicates. The coefficient correlations of the two repeated but independent experiment are 0.994 (virus 1 vs virus 2) and 0.994 (control 1 vs control 2)at 6h postrelease; 0.980(virus 1 vs virus 2) and 0.975(control 1 vs control 2) at 12h postrelease. No significant difference at mRNA level were observed between H-l and mock-infected cells, although the NS1 positive cells was above 60%; 12h after release, the mRNA level of 38 genes changed in infected cells compared with uninfected cultures by the factor of 2.5, among which 29 genes are down-regulated and 9 up-regulated. Classification of these candidates into functional clusters identified mainly genes related to transcriptional regulation, signaling transduction and apoptosis, concerning many important pathways. Owing to the use of synchronized cell and the choice of earlier time points, the amount of genes whose expression are regulated is small, and the genes which are related to the traverse of cell cycle were not detected. Furthermore, the genes involved in immune reaction were also not found.10 of these genes were selected for confirmation by real-time RT-PCR (lightcycler). Using RNA sample either from microarray experiments or from independent experiments, the modulation of gene expression of all of them has been verified and the folds changes are rather close to each other, attesting the high reproducibility and reliability of microarray experiments. The results also showed that at 6h after release, the expression of these genes has been influenced, but to an extent that is below the standards set for selecting genes in microarray experiments, suggesting that NS1 is required to surpass a threshold in order to exert observable effect of virus. Changes of mRNA level of 7 of these verified genes after infection in a time course way were then followed by real-time RT-PCR and the results showed that MYC, DUSP2, DKK1, CEBPD, ARHB, and ID3 which are down-regulated at...
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