Mechanism Of MiRNAs Affected By CD14in RAW264.7under B.melitensis M5-90and B.Melitensis M5-90LPS Stimulation

As a receptor of B.melitensis lipopolysaccharide (LPS), cluster of differentiation antigen14(CD14) is one of an important molecule involving in pro inflammatory cytokine release induced by LPS. MicroRNAs (miRNAs) are short non-coding RNAs that involved in many process of life activity, such as cell proliferation, apoptosis, virus defense and bacterial infections. The study indicated that siRNAs against CD14effectively inhibited LPS-induced tumor necrosis factor alpha, chemokine (C-X-C motif) ligand2, interleukin-6release, and NO production.Based on mCD14knockdown RAW264.7(224.3) which were established by Lei Ming et al in our group, miRNA microarray and qRT-PCR were used to analyze the differential expression of miRNAs in224.3, NC, and RAW264.7; miRNA microarray and qRT-PCR were used to analyze the differential expression of miRNAs in224.3, NC, and RAW264.7, which were stimulated with B.melitensis M5-90LPS, respectively. The potential targets were predicted and subjected to biological processes analysis and Gene Ontology(GO). The mechanism of miR-199a-3p regulating target genes was analyzed with qRT-PCR, Western blot and Dual-Glo(?) Luciferase Assay System in224.3cell infected by B.melitensis M5-90.The results showed that mmu-miR-199a-3p, mmu-miR-199a-5p, and mmu-miR-21-5p were up-regulated in224.3cell; mmu-miR-199a-3p and mmu-miR-199a-5p were up-regulated in224.3cell stimulated by B.melitensis M5-90LPS, however, the increase level of mmu-miR-199a-3p was lower than that of no stimulation condition. The expression of Cbl-b was down-regulated in224.3cell infected by B.melitensis M5-90in both mRNA and protein level. Luciferase reporter assays showed that the luciferase activity was inhibited by target luciferase vector and miR-199a-3p mimics co-transfection, these results indicated that miR-199a-3p can regulate the expression of Cbl-b. These findings may improve our understanding about functional mechanism of miRNAs in the attenuating of LPS-induced damaging immune activation and provide an important theoretical basis for exploring the pathogenesis of brucellosis.