Let-7e Inhibits Tnf-Alpha Expression By Targeting The Methyl Transferase Ezh2 In Denv2-Infected Thp-1 Cells

Tumor necrosis factor (TNF), 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 TNF is currently unknown. Our study showed that the TNF 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 TNF expression, but bioinformatics analysis showed that the enhancer of zeste homolog 2 (EZH2) was the potential direct target of let-7e instead of TNF. 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 TNF expression. In the further study of the regulatory mechanism of EZH2 on TNF expression, we showed that siEZH2 promoted EZH1 and H3K4me3 expression and inhibited H3K27me3 expression. More importantly, we revealed that siEZH2 down-regulated NF-B p65 within the nucleus. These findings indicate that the let-7e/EZH2/H3K27me3/NF-B p65 pathway is a novel regulatory axis of TNF 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 TNF alpha.