| 研究課題/領域番号 |
16K08803
|
|---|
| 研究機関 | 北海道大学 |
|---|
研究代表者 |
Carr Michael 北海道大学, 国際連携研究教育局, 助教 (70769588)
|
|---|
| 研究期間 (年度) |
2016-10-21 – 2019-03-31
|
| キーワード | Virus / Long non-coding RNAs / Flavivirus / JEV / Neuronal cells / Deep sequencing / Transcriptomics / Pathogenesis |
|---|
| 研究実績の概要 |
We initially optimised the infection of SH-SY5Y neuronal cells with low and high neurovirulence Japanese encephalitis virus (JEV) strains by testing different multiplicity of infection (MOI) and time-points by fluorescence-activated cell sorting and immunofluorescence microscopy with an anti-JEV polyclonal antibody. The cellular transcriptomic response, specifically focusing upon long non-coding RNAs (lncRNAs) and protein-coding genes, expressed upon JEV infection of SH-SY5Y neuronal cells was then investigated by HiSeq next-generation sequencing with >10 million reads per each biological replicate.
We have identified within these datasets by gene ontology analysis of infected and mock-infected neuronal cells that there is a significant upregulation of both endoplasmic reticulum (ER) signaling pathway and ER unfolding protein response (UPR) stress-response genes (e.g. DDIT3, HERPUD1, ATF3, ASNS, DNAJB9, HSPA5), together with a selection of previously uncharacterised lncRNAs. We have validated the upregulation of ER stress (exemplified by DDIT3) and all deregulated lncRNAs in neuronal and other cell types by qRT-PCR analysis and confirmed a significant upregulation of both groups of transcripts 24 hours post-infection.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Since obtaining the Kakenhi grant in November 2016, in the previous six months we have conducted our first next-generation sequencing run and successfully identified lncRNAs deregulated in neuronal cells in experiments with two different JEV strains using a bioinformatics pipeline. We have validated by qRT-PCRs specific to these lncRNAs that the transcripts are deregulated in virus-infected neuronalcells and, thus, we have potential candidates to experimentally investigate.
|
| 今後の研究の推進方策 |
We have obtained siRNAs targeting the lncRNAs that were identified to be upregulated upon JEV infection and optimisation of the siRNA transfection of the SH-SY5Y (and other neuronal cell lines) is proceeding. Once established, the lncRNA knockdown cells will be infected with JEV strains and the roles of these RNA transcripts in JEV replication examined by qRT-PCR and immunoblotting.
An additional next-generation sequencing run employing RNA derived from mock-infected and virally-infected neuronal cells from samples with higher multiplicity of infections, more than 3 biological replicates per experimental condition and >20 million reads per sample is also planned to determine if additional lincRNA targets can be identified for validation.
|
| 次年度使用額が生じた理由 |
Kakenhi was received in November of FY2016 so only six months has been available to spend research funding and not a full financial year.
|
| 次年度使用額の使用計画 |
Increased expenditure is projected with regards to validation of the experimental targets and also for additional next-generation sequencing runs to identify further candidates for validation.
|
