Two-layer regulation of TRAF6 mediated by both TLR4/NF-kB signaling and miR-589-5p increases proinflammatory cytokines in the pathology of severe acute pancreatitis.

Inflammation is a leading cause of severe acute pancreatitis (SAP). MicroRNAs (miRNAs) are emerging as important regulators involved in the pathogenesis of many diseases including pancreatitis. To identify miRNAs that contribute to the pathology of SAP, we carried out a miRNA-specific microarray analysis using the biopsies donated by SAP patients. We totally obtained 50 differentially expressed miRNAs, including 20 upregulated and 30 downregulated miRNAs, respectively. We focused our current study on revealing the downstream target and the upstream regulatory mechanism of miR-589-5p, the most downregulated miRNA in our candidate lists. Our prediction results indicated that miR-589-5p might target TRAF6 (tumor necrosis factor receptor-associated factor 6), a critical member of the TLR4/NF-kB (Toll-like receptor 4/nuclear transcription factor-kB) pathway. Using different strategies such as in vitro overexpression or downregulation of miR-589-5p and treatment with lipopolysaccharide (LPS), we found that the expression of TRAF6 was regulated by two-layer mechanisms. On the one hand, TRAF6 was transcriptionally controlled by a DNA methylation mediated downregulation of miR-589-5p. On the other hand, the activation of TLR4/NF-kB signaling also could increase the protein level of TRAF6. The increased TRAF6 aggravated the downstream signaling and caused the translocation of NF-kB subunits from the cytoplasm to the nucleus, where NF-kB transcription factors induced the expression of proinflammatory cytokine genes. The maturation and production of proinflammatory cytokines induced inflammatory response and caused the occurrence of SAP.