Effects of microRNA-294 on inflammatory factor of sepsis by targeting triggering receptor expressed on myeloid cells-1 / 中华危重病急救医学

ABSTRACT

Objective To investigate the effects of microRNA-294 (miR-294) on tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6) and high mobility group box 1 (HMGB 1) secretion in sepsis by targeting triggering receptor expressed on myeloid cell-1 (TREM-1).Methods miRNA-294 was predicted to regulate TREM-1 specially through bioinformatics analysis.Mice macrophage cell lines RAW264.7 were cultured in vitro,the cells were divided into non-inflammatory stage and inflammatory stage,and the cells in the two stages were subdivided into five groups as followsnormal control (NC),NC mimic transfection (NCm),NC inhibitor transfection (NCi),miR-294 mimic transfection (miR-294m) and miR-294 inhibitor transfection (miR-294i) groups.The ability of miR-294 was confirmed with dual-luciferase activity assay.At non-inflammatory stage,the cells were transfected with mimic or inhibitor of miR-294 or NC using TurboFectTM siRNA Transfection Reagent for 48 hours,mRNA expression of TREM-1 was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR).At inflammatory stage,6 hours after stimulation by lipopolysaccharide (LPS,1 mg/L),the concentrations of TNF-α,IL-6 and HMGB1 were determined by enzyme linked immunosorbent assay (ELISA),the protein expression of TREM-1 was determined by Western Blot.Results ① Dual-luciferase activity assay demonstrated that TREM-1 was the target of miR-294.② In non-inflammatory stage,the expression of TREM-1 mRNA (2-ΔΔ~) in miR-294m group was significantly lower than that of the NC and NCm groups (0.673 ± 0.049 vs.1.000 ± 0.003,0.915 ± 0.039,t1=2.184,t2=5.421,both P＜0.001),the expression of TREM-1 protein (gray scale) was (50.00 ± 1.19)％ of NCm group (t=41.586,P＜0.001).③ In inflammatory stage,the concentrations of TNF-α (ng/L) in miR-294m group was significantly lower than that of the NC group (1 547.18 ±47.18 vs.2 702.11 ± 327.20,t=4.212,P=0.010),the concentrations of IL-6 (ng/L) was significantly lower than that of the NC and NCm groups (505.28 ± 33.33 vs.837.66 ± 69.43,918.72 ± 119.39,t1 =4.382,P1=0.015; t2=5.451,P2=0.021),the level of TREM-1 protein (gray scale) was (51.33 ±0.88)％ of NCm group (t=63.368,P＜0.001).Conclusion miR-294 reduce TNF-α and IL-6 secretion in LPS-induced RAW264.7 through inhibiting the expression of TREM-1 specifically.