title: [Effects of microRNA-294 on inflammatory factor of sepsis by targeting triggering receptor expressed on myeloid cells-1].
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 (HMGB1) 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 follows: normal control (NC), NC mimic transfection (NCm), NC inhibitor transfection (NCi), miR-294 mimic transfection (miR-294 m) 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 TurboFect™ 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(-ΔΔCt) ) 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-294 m 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).
  
  
  CONCLUSIONS
  miR-294 reduce TNF-α and IL-6 secretion in LPS-induced RAW264.7 through inhibiting the expression of TREM-1 specifically.
authors.affiliation: Department of Anesthesiology, the Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, Guangdong, China, Corresponding author: Zhang Liangqing, Email: zhanglq1970@163.com.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Liu
authors.forename: Dianqing
authors.initials: D
authors.lastname: Cao
authors.forename: Guixi
authors.initials: G
authors.lastname: Mo
authors.forename: Liangqing
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000818:Animals; D008070:Lipopolysaccharides; D051379:Mice; D035683:MicroRNAs; D022423:Myeloid Cells; D000067996:RAW 264.7 Cells; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018805:Sepsis; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha
chemical_list: D008070:Lipopolysaccharides; D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha