Dynamic changes in global microRNAome and transcriptome reveal complex miRNA-mRNA regulated host response to Japanese Encephalitis Virus in microglial cells.

Bharti Kumari,Anirban Basu,Arup Banerjee,Ashish Chandra Trivedi,Pratistha Jain,Sudhanshu Vrati,Shaoli Das,Suman Ghosal,Jayprokas Chakrabarti,Bibhabasu Hazra

doi:10.1038/srep20263

Abstract

Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation.

Highlights

MicroRNAs have been emerged as a powerful tool to regulate gene expression through the RNA interference pathway
Cells were infected at high multiplicity of infection (MOI = 5) to enhance infection probability and improve signal to noise ratio and infected cells were washed with 1× PBS after virus adsorption
The experiment was done in triplicate, Virus infection and replication was monitored at three different time points

Summary

Introduction

MicroRNAs (miRNAs) have been emerged as a powerful tool to regulate gene expression through the RNA interference pathway. Using an Affymetrix microarray platform, we have profiled global cellular miRNA and mRNA expression in human microglial (CHME3) cells infected with JEV at multiple time points (6, 24, and 48 h). These time points have afforded us a view of the miRNAs and transcriptomic changes occurring both before and during viral replication. Compared to more traditional microarray platforms, the Affymetrix platform combines customizable arrays on a single chip, with each array containing probes for human pre-miRNA, mature miRNA, small nucleolar RNA (snoRNA) and small Cajal body specific RNA (scaRNA) as annotated in the Sanger miRBase 17. We have provided evidence suggesting that JEV infection induces NOTCH signaling pathway activation, down regulating several miRNAs that target multiple genes in NOTCH pathway. Over expression of these down-regulated miRNAs have attenuated JEV induced pro-inflammatory cytokine production

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