| The immunopathology of dengue virus (DENV) infection is associated with increased TNF-alpha production. In this study, small RNA-mediated regulation of TNF-alpha and the effect of TNF-alpha knockdown during DENV infection were analyzed. This provides insight into the role of TNF-alpha during DENV infection, both in terms of its contribution to immunopathogenesis and its regulatory mechanism by miRNAs. Utilizing a lentiviral expression system, human monocytic U937 cells that express short hairpin RNAs designed to target TNF-alpha mRNA were established. TNF-alpha expression was downregulated in these monocytes, and upon DENV infection they showed decreased endothelial cell activation ability. This demonstrates an overall decrease in the proinflammatory response upon TNF-alpha knockdown during DENV infection. To analyze the role of microRNAs (miRNAs) in the TNF-alpha response, miRNAs that potentially target the 3' UTR of TNF-alpha were predicted. Many of the miRNAs were differentially regulated during DENV infection. miR-320a and miR-592 were among those downregulated, and chosen for further analysis. TNF-alpha post-transcriptional regulation by miR-320a and miR-592 was confirmed utilizing a TNF-alpha 3'UTR luciferase reporter. U937 cells transfected with miR-320a and miR-592 mimics followed by DENV infection displayed decreased TNF-alpha expression and their culture supernatants demonstrated decreased ability to activate endothelial cells. It is concluded that one function of these miRNAs is to negatively regulate TNF-alpha, and their downregulation contributes to the immunopathogenesis of DENV infection. |
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