Project description:Japanese encephalitis virus (JEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in humans. Survivors of this infection often develop life-long neurological sequelae. Short-chain fatty acids (SCFAs) produced in the gut are vital mediators of the gut-brain axis. We aimed to study microRNA-based mechanisms of SCFAs in an in vitro model of JEV infection. N9 microglial cells were pre-treated with SCFA cocktail before JEV infection. Cytokine bead analysis (CBA), immunoblotting and PCR were performed to analyse relevant inflammatory markers. microRNA sequencing was performed using Illumina Hiseq and Bioinformatical tools were used for differentially expressed (DE) miRNAs and Weighted gene co-expression network analysis (WGCNA). microRNA mimic/inhibitor experiments and luciferase assay were performed to study miRNA-target interaction. A significant reduction in MCP1 and TNFα along with reduced expression of phospho-NF-κB was observed in SCFA conditions. Significant attenuation of HDAC activity and protein expression was recorded. miRNA sequencing revealed 160 DE miRNAs in SCFA+JEV treated cells at 6 hours post infection (HPI). WGCNA revealed miR-200a-3p, a hub miRNA significantly upregulated in SCFA conditions. Transcription factor ZBTB20 was bioinformatically predicted and validated as a gene target for miR200a-3p. Further miRNA mimic/inhibitor assay demonstrated that miR-200-3p regulated ZBTB20 along with Iκβα that possibly dampened NF-κB signal activation downstream.
Project description:In order to further determine the je virus (JEV) infection in C57BL / 6 mice after its miRNA levels change, we use the Agilent micrornas chip as a platform, to detect the uninfected group (control group) and the experimental mouse total RNA, it is found that the miRNA levels decrease infection group, and picked out the ten changes obviously micrornas verification, the result is the same.
Project description:B16F10, a murine melanoma cel line is cytolytic to JEV infection. We have identified a B16F10 cell line variant which is non-cytolytic to JEV infection. This variant cell line has two types of cells, one which is persistently infected JEV and another which is resistant to JEV infection. The JEV-resistant cells (B16F10r) were seperated from the presistently infected cells by single cell cloning. To understand the mechanism of JEV resistance microarray analysis was caried out to Identify and characterize genes differentially expressed during in uninfected/JEV-infected B16F10 and B16F10r cells.
Project description:B16F10, a murine melanoma cel line is cytolytic to JEV infection. We have identified a B16F10 cell line variant which is non-cytolytic to JEV infection. This variant cell line has two types of cells, one which is persistently infected JEV and another which is resistant to JEV infection. The JEV-resistant cells (B16F10r) were seperated from the presistently infected cells by single cell cloning. To understand the mechanism of JEV resistance microarray analysis was caried out to Identify and characterize genes differentially expressed during in uninfected/JEV-infected B16F10 and B16F10r cells. Agilent one-color experiment,Organism: Mus musculus ,Agilent Custom Mouse Whole Genome Mouse 8x60k Gene expression designed by Genotypic Technology Private Limited, Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:Japanese encephalitis virus (JEV) is the leading cause of epidemic encephalitis worldwide. JEV-induced neuroinflammation is characterized by profound neuronal cells damage accompanied by activation of glial cells. Albeit long non-coding RNAs (lncRNAs) have been emerged as important regulatory RNAs with profound effects on various biological processes, it is unknown how lncRNAs regulate JEV-induced inflammation. Here, using microarray approach, we identified 618 lncRNAs and 1007 mRNAs differentially expressed in JEV-infected mice brain.
