| In order to screen related genes of rotavirus and its host interaction, the rotavirus strain CC0812-1 isolated in our laboratory was inoculated to intestinal epithelial HT29 with a titer of 1 TCID50/cell, total RNAs were extracted from RV infected HT29 cells at 12 hpi. Transcriptome sequence of RV infected HT29 cells was conducted by Illumina HiSeq 2500. Comparing to transcriptome of HT29 cells, totally 473 differential expression genes were screened, of which 418 genes were up-regulated and the other 55 genes were down-regulated,82 genes were annotated to 12 immune-related signaling pathways, which were widely involved in pathogen-associated molecular pattern recognition, regulation of complement system, cytokine reaction, apoptosis and other anti-viral response related signaling process through KEGG analysis. In order to verify the reliability of RNA-Seq technology for transcriptome sequence, seven differential expression genes were randomly choosed and quantified by qRT-PCR. The variation tendency of these genes detected by qRT-PCR is consistent with that by RNA-Seq analysis in copy numbers, which indicated the RNA-Seq technology was a feasible method in screening differential expression genes. These 473 differential expression genes screened by RNA-Seq might be potential targets for rotavirus and its host interaction, and rotavirus innate immune.To investigate the mechanism of rotavirus up-regulating IFN λ1 mRNA, rotavirus CC0812-1 was inoculated to intestinal epithelial cells HT29 and HCT116 with a titer of 1 TCID50/cell, Total RNA extracted from cells was extrated from RV infected cells at 6 hpi, 12 hpi, and 24 hpi, respectively and then subjected to the qRT-PCR for the mRNA levels of IFN λ1. The results showed that RV infection up-regulate IFN λ1 mRNA expression in time-dependent mode, IFN λ1 mRNA expression began to up-regulation at 6 hpi (11.54 fold for HT29 and 4.95 fold for HT116), reached the peak at 12 h pi (82.56 fold for HT29 and 43.41 fold for HT116) and decreased at 24 hpi (66.76 fold for HT29 and 22.83 fold for HT116). Up-regulation of IFN λ1 mRNA induced by RV infection is also titers-dependent, higer the titer of RV inoculated to HT29 and HCT116 cells, higer the expression level of IFN λ1 mRNA. IFN λ1 mRNA expression at 12 hpi was upregulated 2.03 fold for HT29 and 2.02 fold for HT116 with a titer of 0.01 TCID50/cell,10.29 fold for HT29 and 11.59 fold for HT116 with a titer of 0.1 TCID50/cell and 82.56 fold for HT29 and 43.41 fold for HT116 with a titer of 1 TCID50/cell, respectively. Meanwhile, mRNA expression of pattern recognition receptor RIG-I and MDA5 and the major interferon regulatory factor (IRF1, IRF3, IRF7, IRF9) were quantified by qRT-PCR, mRNA expression of RIG-I was upregulated 5.30 fold for HT29 and 2.35 fold for HT116, MDA5 was upregulated 5.67 fold for HT29 and 2.20 fold for HCT116 IRF1 was upregulated 4.96 fold for HT29 and 2.32 fold for HT116, IRF3 was upregulated 1.72 fold for HT29 and 1.49 fold for HT116, IRF7 was upregulated 3.61 fold for HT29 and 2.52 fold for HT116, IRF9 was upregulated 1.5 fold for HT29 and 1.64 fold for HT116 with a titer of 1 TCID50/cell at 12 hpi. RNAi results showed that IFN λ1 mRNA expression in HCT116 cells induced by RV infection decreased 0.37 times when RNA intefering RIG-I, decreased 0.73 times when RNA intefering MDA5, and decreased 0.27 times when RNA intefering both RIG-I and MDA5, respectively. Thus, It is suggested that rotavirus might upregulated IFN λ1 mRNA expression through RIG-I/MDA5 pathway. |
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