DENV-NS1 drives novel microRNAs to disrupt cerebral endothelial barrier functions.

Dengue virus non-structural protein 1 (DENV-NS1) plays a key role in the vascular leakage associated with severe dengue; however, the molecular mechanisms by which NS1 induces endothelial dysfunction remain poorly understood. This study aimed to identify a new aspect of the pathogenic strategy: the post-transcriptional reprogramming of host endothelial cells via microRNAs (miRNAs) in NS1-induced DENV pathogenesis. Small RNA sequencing and subsequent bioinformatic analysis identified novel miRNA-gene regulatory networks responsive to DENV-NS1 in human brain microvascular endothelial cells. Multiple dysregulated novel miRNAs were identified as potential regulators of critical antiviral components (STAT1, SOCS1-6, OAS1), suggesting suppressed interferon signaling. Downregulation of identified novel miRNAs targeting IL-6, IL-10, IL-1α, IL-17A, CXCR4, and CXCR5 indicates increased pro-inflammatory cytokine production and chemokine-driven immune cell recruitment, thereby worsening endothelial inflammation and vascular leakage. The simultaneous targeting of cytoskeletal regulators (RAC1, ROCK1/2) and tight junction proteins (CLDN1, CLDN2, CLDN16, CLDN22) suggests a two-phase disruption of endothelial stability, characterized by cytoskeletal remodeling and compromised tight junction integrity as a bystander effect of DENV-NS1. The downregulation of several miRNAs targeting XIAP, MDM2/4, and SMAD family genes established the involvement of novel miRNAs in apoptosis regulation and TGF-β/SMAD-mediated endothelial cell signaling. Our findings suggest that DENV-NS1 alters endothelial miRNA networks, leading to reduced antiviral immunity, increased inflammation, and compromised vascular integrity. This study identifies novel miRNA-mediated regulatory nodes that could function as therapeutic targets to prevent or mitigate dengue-associated vascular leakage.