Dengue hemorrhagic fever (DHF)/Dengue shock syndrome (DSS) is a fatal infectious disease that demands an effective treatment. Interferon (IFN)-stimulated genes (ISGs) induced by dengue virus (DENV) exert antiviral effects. Among ISGs, IFN-α inducible gene 6 (IFI6) was increased in DENV infected human umbilical vascular endothelial cells (HUVECs) by microarray analysis in our previous study. However, its function is incompletely understood. In this study, we confirmed that IFI6 was markedly induced in DENV infection of both primary HUVECs and EA.hy926 cell lines. Recombinant EA.hy926 cell lines in which IFI6 was either over-expressed (IFI6+/+) or knocked-down (IFI6-/-) were generated. The activation of caspase-3 and intrinsic apoptosis-related protein caspase-9 were down-regulated in IFI6+/+ but up-regulated in IFI6-/- cells at 24–48 hrs post-infection. After incubation with DENV for 48 hrs, the mitochondrial membrane potential (Δψ(m)) was more stable in IFI6+/+ cells but reduced in IFI6-/- cells, as assayed by fluorescence staining with JC-1. We observed that Bcl-2 expression was increased in IFI6+/+ and decreased in IFI6-/- cells. By contrast, Bax expression was decreased in IFI6+/+ and increased in IFI6-/- cells. It is presumed that the anti-apoptotic function of IFI6 is expressed by regulating the rheostatic balance between bcl-2/bax expression and inhibition of Δψ(m) depolarization during DENV infection of vascular endothelial cells(VECs). In addition, the pro-apoptotic protein X-linked Inhibitor of Apoptosis (XIAP)-Associated Factor 1(XAF1) expression had been reported to be up-regulated and led to the induction of apoptosis in DENV2-infected VECs,but the relationship between XAF1 and IFI6 dengue virus-induced apoptosis in VECs warrants further study.

Dengue is the most prevalent mosquito-borne viral disease in tropical regions. Severe cases may progress to Dengue hemorrhagic fever, suggesting vascular endothelial dysfunction in disease pathogenesis. In our previous study, we found that Dengue virus type 2 (DENV2) induced apoptosis of vascular endothelial cells via FasL/Fas- and XIAP-associated factor 1 (XAF1)-dependent pathways. In this paper, we demonstrate that DENV2 can induce autophagy in primary human umbilical vein endothelial cells (HUVECs) and the human umbilical vein endothelial cell line EA.hy926. Inhibition of autophagy with 3-methyl adenine promoted apoptosis, while inhibition of apoptosis with Z-VAD-FMK facilitated autophagy in DENV2-infected HUVECs and EA.hy926 cells. Interferon-alpha-inducible protein 6 (IFI6), a putative apoptosis regulator, inhibited DENV2-induced autophagy in EA.hy926 cells, while XAF1, an inhibitor of anti-apoptotic XIAP, facilitated autophagy. Molecular regulators of apoptosis and autophagy interact at multiple levels to determine cell fate. Our data suggest that XAF1 and IFI6 are involved in regulating the balance between autophagy and apoptosis in DENV2-infected endothelial cells.

Tumor necrosis factor α (TNFα), an important inflammatory cytokine, is associated with dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), a severe pathological manifestation of dengue virus (DENV) infection. However, the regulatory mechanism of microRNA on TNFα is currently unknown. Our study showed that the TNFα expression increased immediately and then later decreased, while a marked increase for the miRNA let-7e was detected in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMCs). From this study, we found that let-7e was able to inhibit TNFα expression, but bioinformatics analysis showed that the enhancer of zeste homolog 2 (EZH2) was the potential direct target of let-7e instead of TNFα. EZH2 methyl transferase can produce H3K27me3 and has a negative regulatory role. Using a dual-luciferase reporter assay and Western blotting, we confirmed that EZH2 was a direct target of let-7e and found that siEZH2 could inhibit TNFα expression. In the further study of the regulatory mechanism of EZH2 on TNFα expression, we showed that siEZH2 promoted EZH1 and H3K4me3 expression and inhibited H3K27me3 expression. More importantly, we revealed that siEZH2 down-regulated NF-κB p65 within the nucleus. These findings indicate that the let-7e/EZH2/H3K27me3/NF-κB p65 pathway is a novel regulatory axis of TNFα expression. In addition, we determined the protein differences between siEZH2 and siEZH2-NC by iTRAQ and found a number of proteins that might be associated with TNFα.