2017 Fiscal Year Research-status Report
Long Non-Coding RNAs in Viral Infection
| Project/Area Number |
16K08803
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| Research Institution | Hokkaido University |
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Principal Investigator |
Carr Michael 北海道大学, 国際連携研究教育局, 助教 (70769588)
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| Project Period (FY) |
2016-10-21 – 2019-03-31
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| Keywords | Virus / Long non-coding RNAs / Flavivirus / JEV / Neuronal cells / Deep sequencing / Transcriptomics / Pathogenesis |
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| Outline of Annual Research Achievements |
We have conducted a much deeper second next-generation sequencing run of virus-infected cells employing the high-throughput NovaSeq 6000 platform. Japanese encephalitis virus (JEV) infected and uninfected SH-SY5Y neuroblastoma cells 24 hours post-infection (hpi) were examined by a paired-end 150 bp RNA-seq strategy on the NovaSeq 6000 platform (Illumina) with an output of 110 Gb and approximately 40 million reads per biological replicate (n=4 biological replicates for both the treated and untreated conditions). Bioinformatic analyses were performed with edgeR and DESeq2 software and gene ontology (GO) term analysis was employed to identify deregulated pathways. Differentially expressed targets were validated by RT-qPCR at 0 hpi, 8 hpi and 24 hpi.
We have thus determined the transcriptomic response - differentially expressed protein-coding genes and long non-coding RNA (lncRNA) transcripts - in human neuronal cells following JEV infection. This dataset provides a basis for examining the pathology associated with flavivirus replication in neurons. GO term analysis of infected and uninfected SH-SY5Y neuroblastoma cells 24 hpi showed upregulation of endoplasmic reticulum (ER) stress signalling during JEV infection. The top two GO terms were ER unfolded protein response and ER-nucleus signalling (both FDR 3.1 x 10-15) is consistent with flavivirus replication in the ER. Specific lncRNA and protein-coding genes of interest from other pathways have been investigated and demonstrated to be significantly deregulated upon JEV infection of SH-SY5Y cells.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In the last financial year, we have conducted our second next-generation sequencing run employing the high-throughput NovaSeq 6000 platform, with more biological replicates and with far greater sequencing depth than the previous next-generation sequencing run, i.e. 40 million reads each for four biological replicates for both the treated and untreated experimental conditions. Transcriptomic analyses indicate upregulation of ER stress signalling during JEV infection of neuronal cells in vitro. Promising candidate lncRNA and deregulated (up- and down-regulated) transcripts from other pathways are being examined for their effect once perturbed on JEV replication in neuronal cells.
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| Strategy for Future Research Activity |
Future work will involve completing the testing of the effects of knockdown and overexpression of these up- and down-regulated candidates and further functional characterisation work to ascertain whether these transcripts are directly implicated in JEV replication in neurons. Manuscript preparation will begin before the end of the financial year for dissemination of the research findings in international peer-reviewed journals.
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| Causes of Carryover |
In the previous financial year, access to the NovaSeq6000 platform was an important matter. This has now been resolved so that validation of the targets identified by RNA-seq to be deregulated in neurons upon JEV infection can be tested experimentally. Reagents to validate these targets will utilise the remaining funds, as planned in the original budget proposal.
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