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 follows:normal 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.

To carry out pathologic diagnoses and whole-genome sequence analyses of the Jaagsiekte sheep retrovirus (JSRV) in Xinjiang, China, we first observed sheep suspected to have the JSRV. Then, the extracted virus suspension was observed by transmission electron microscopy (TEM). Total RNAs from lungs of JSRV-infected sheep were extracted and reverse-transcribed using a cDNA synthesis kit. Six pairs of primers were designed according to the exogenous reference virus strain (AF105220). Reverse transcription-polymerase chain reaction was carried out from JSRV-infected tissue, and the whole genome of the JSRV sequenced. Our results showed: flow of nasal fluid ("wheelbarrow test"); different sizes of adenoma lesions in the lungs; papillary hyperplasia of alveolar epithelial cells; alveolar cavity filled with macrophages; dissolute nuclei in central lesions. TEM revealed JSRV particles with a diameter of 88 nm to 125. 4 nm. The full-length of the viral genome sequence was 7456 bp. BLAST analyses showed nucleotide homology of 96% and 95% compared with that of the representative strain from the USA (AF105220) and UK (AF357971). Nucleotide homology was 89.8% and 89.9% compared with the endogenous Jaagsiekte sheep retrovirus, Inner Mongolia strain (DQ838493) and USA strain (EF680300). The specific pathogenic amino-acid sequence "YXXM" was found in the TM district, similar to the exogenous JSRV: this gene has been reported to be oncogenic. This is the first report of the complete genomic sequence of the exogenous JSRV from Xinjiang, and could lay the foundation for study of the biological characteristics and pathogenic mechanisms of the pulmonary adenomatosis virus in sheep.
Amino Acid Sequence ; Animals ; China ; Genome, Viral ; Jaagsiekte sheep retrovirus ; classification ; genetics ; isolation & purification ; pathogenicity ; Lung ; pathology ; virology ; Molecular Sequence Data ; Phylogeny ; Pulmonary Adenomatosis, Ovine ; pathology ; virology ; Sheep ; Viral Proteins ; chemistry ; genetics ; Virulence