expression level was dramatically suppressed upon LPS-stimulation and showed a time-dependent manner (Figure 3A)

expression level was dramatically suppressed upon LPS-stimulation and showed a time-dependent manner (Figure 3A). negative regulator of inflammatory response by targeting NF-B1 (p50). is probably one of the most studied miRNA that regulates inflammatory response by targeting TRAF6 and IRAK1 following lipopolysaccharide (LPS)-stimulation [11]. can target TNF- resulting in inhibition of inflammatory response [12]. and regulate inflammation responses by targeting IL-1 respecting TAB2 and SOCS1 [13,14]. However, it remains largely unknown as to how inflammation is regulated by miRNA in immune response. is the homologue of human is a member of family, also known as family [16,17]. It is involved in the regulation of cell proliferation, cell differentiation, diabetes and male infertility [15,18C20]. In our preliminary study, was predicted to target several sites of inflammatory factors using the software programs. Little is known about the involvement of during inflammatory response. RAW264.7 was a mouse peritoneal macrophage cell line established from a tumour induced by Abelson murine leukaemia virus. It is one of the commonly used inflammatory cell models. Here, we found that the level of was down-regulated in RAW264.7 cells by administration of LPS. We also showed that mimic transfection resulted in an inhibition in pro-inflammatory cytokines mRNA expression, such as IL-1, IL-6, TNF- and bHLHb21 increased anti-inflammatory cytokines IL-4 and IL-10 expression. Besides, NF-B1 (p50) was identified as a functional target, through which acted as a negative regulator in macrophage inflammatory response. Moreover, may promote cell-cycle procession and cell proliferation. Our findings demonstrate that the level of is down-regulated by LPS-stimulation and is a negative regulator of the immune response. Materials and methods RAW264. 7 cells culture and treatment RAW264.7 was a mouse peritoneal macrophage cell line established from a tumour induced by Abelson murine leukaemia virus. It is one of the commonly used inflammatory cell models. Cells were cultured in DMEM (Hyclone) medium supplemented with 10% FBS at 37C in 5% CO2. RAW264.7 cells were seeded in six-well plates at a density of 2 105 cells/well. Twenty four hours later, the cell medium was replaced with fresh medium. Cells were collected at 0, 2, 4, 8, 12 and 24 h after 1 g/ml LPS (SigmaCAldrich, U.S.A.) induction. mimics transfection mimics and inhibitors were purchased from GenePharma (China). RAW264.7 cells were seeded into six-well plates for 12 h. The cells were replaced with fresh medium (DMEM + 10% FBS) and transfected with 50 nM mimics and inhibitors using Lipofectaime 2000 (Invitrogen TM, U.S.A.) according to the manufacturers instructions. After transfection for 24 h, the medium was replaced with fresh medium containing 1 g/ml LPS. The WEHI-345 cells were collected after LPS induction for 8 h. Quantitative real-time PCR Total RNA was extracted from treated cells with TRIzol (Invitrogen) according to the instructions of the manufacturer. For mRNA analysis, reverse transcription was performed using a first-strand cDNA synthesis kit (Toyobo, Japan). To quantify mature expression, a commercial Bulge-Loop? miRNA quantitative reverse transcription detection WEHI-345 method was used with and the endogenous control gene were from RiboBio (China), whereas other primers were designed by the Primer Express software and synthesized from Invitrogen (Table 2). Fold change was calculated using the 2?inhibitorsUUCAAAACAUGAAUUGCUGCUGInhibitors NCCAGUACUUUUGUGUAGUACAAmimicsSense: CAGCAGCAAUUCAUGUUUUGAAAntisense: CAAAACAUGAAUUGCUGCUGUUMimics NCSense: UUCUCCGAACGUGUCACGUTTAntisense: ACGUGACACGUUCGGAGAATTstem loop-primerGTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTCAAAin the TLR signalling pathways. Then, the miRNA-binding sites in target genes and the binding free energy were analysed and calculated on the website (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) [21]. Luciferase reporter assays 293T cells were cultured in DMEM medium and seeded in six-well plates at a density of 2 105 cells/well. The 3-UTRs of mouse NF-B1 (p50) and their corresponding WEHI-345 mutated 3-UTRs were amplified by PCR using the primers shown in Table 1 and cloned into psiCheck-2 dual-luciferase reporter vector (Promega). Co-transfection was performed with constructed plasmid, miRNA mimics or inhibitors using Lipofectamine 2000. luciferase activities were measured using the Dual-Luciferase Reporter Assay.