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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. [ABSTRACT FROM AUTHOR]