PDF《International Immunopharmacology》

100 μg/ml streptomycin) at

37 °C in 5% CO2. The primary mouse peritoneal macrophages was isolated and maintained as de- scribed [9]. Cells were seeded into 6-well plate and incubated overnight. Lipofectamine?

2000 (Invitrogen, Shanghai, China) transfection re- agent was used for transfection of plasmid and miR-709 mimics (RIBOBIO, Guangzhou, China), miR-709 inhibitors (RIBOBIO, Guang- zhou, China), Negative Control (RIBOBIO, Guangzhou, China), Inhibitor International Immunopharmacology

36 (2016) 333C338 ? Corresponding author at: School of Life Sciences, Sun Yat-sen University, North Third Road, Guangzhou Higher Education Mega Center, Guangzhou, Guangdong 510006, PR China. E-mail address: [email protected] (D. Mo). http://dx.doi.org/10.1016/j.intimp.2016.04.005 1567-5769/?

2016 Published by Elsevier B.V. Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Control (RIBOBIO, Guangzhou, China) according to the manufacturer'

s instructions. 2.3. RNA extraction and quantitative real-time RT-PCR (qPCR) Total RNAs were extracted with Trizol (Invitrogen) and treated with DNase I (Fermentas, Guangzhou, China). Isolated total RNAs were tran- scribed with Oligo (dT) or Stem-loop reverse primers by using Reverse Transcription System (Promega). qPCRs were performed with gene- speci?c primers and SYBR Premier Dimer Eraser (Takara, Dalian, China) on the LightCycler

480 (Roche, Basel, Switzerland). The relative mRNA expression level of each gene was normalized against house- keeping gene GAPDH. The relative expression of miRNA was normalized against U6 snRNA. The ΔC method was used to calculate the data. 2.4. Dual luciferase assay HEK 293T cells were cultured on 48-well plates and transfected with a mixture containing

50 ng psiCHECK-2 recombination vector and miR-

709 mimics or negative control using Lipofectamine?

2000 transfection reagent. Cell culture medium was changed

6 hours post transfection, and cells were assayed

48 h later, using Dual-Glo luciferase system (Promega) and measured with a BioTek? Synergy?

2 Multi-mode Mi- croplate Readers (BioTek, Vermont, USA). 2.5. Immunoblotting Cells were collected and lysed using cell lysis buffer (Beyotime, Shanghai, China) supplemented with complete protease inhibitor cock- tail (Sigma, Shanghai, China) and phosphatase inhibitors (5 mM Na4P2O7,

50 mM NaF,

1 mM vanadate). Equal loading of total proteins were assured using a Pierce BCA Protein Assay Kit,

20 μg of total protein per sample was loaded in each well of the SDS-PAGE gels. After transfer, the membrane was blocked with TBS-Tween20 containing 5% skimmed milk, the membrane was incubated with rabbit monoclonal anti-GSK- 3β (#9315, CST), anti-β-catenin (#8480), anti-GAPDH (sc-59540, Santa Cruz Biotechnology, Shanghai, China) for

1 h at room temperature and then incubated with secondary antibody (#7074s, CST) at room temperature for

1 h, then the proteins were detected by an enhanced chemiluminescence detection kit (Thermo Scienti?c, Guangzhou, China). 2.6. Statistical analysis All data are expressed as mean ± standard deviation (SD) based on at least three experiments. Statistical analysis was determined by using Student'

s two-tailed t-test. The values of P b 0.05 was considered to be statistically signi?cant. 3. Results 3.1. MiR-709 is up-regulated by LPS in murine macrophages To identify potential miRNA involved in innate immunity, we previ- ously preformed a high-throughput microarray assays. The microarray pro?le revealed that miR-709 was remarkably up-regulated in spleen tissue from LPS-treated mouse. In this study, we further evaluated the effects of miR-709 on the regulation of in?ammation. To determine whether LPS-induced increase in the expression of miR-709 was also observed in macrophages, murine primary peritoneal macrophage cells and murine macrophage cell line RAW264.7 cells were employed to investigate the effect of LPS treatment on miR-709 expression. We stimulated these macrophage cells with LPS at different concentrations and miRNA expression was measured by qPCR. As shown in Fig. 1A and C, miR-709 was signi?cantly up-regulated in LPS stimulated RAW264.7 cells and murine primary peritoneal macrophage cells in a dose- dependent pattern. We also treated these cells with LPS for different time. The expression of miR-709 was time dependently up-regulated in primary peritoneal macrophage cells and RAW264.7 cells (Fig. 1B and D). Thus we suggest that murine macrophage miR-709 may be in- volved in the modulating of LPS-induced in?ammation in murine macrophages. 3.2. MiR-709 modulates LPS-induced in?ammation To investigate the biological functions of miR-709 in LPS-induced in- ?ammation, we transfected RAW264.7 cells with miR-709 mimics or mimics control and measured the production of pro-in?ammatory cyto- kines. As shown in Fig. 2CCF, overexpression of miR-709 signi?cantly inhibited TNF-α, IL-6 and IL-1β production at both the mRNA and the protein level following LPS treatment of RAW264.7 cells. This suggested that miR-709 plays an inhibitory role in LPS-induced macrophage cyto- kine production. To further investigate this, we measured the cytokine production of RAW264.7 cells with miR-709 knockdown following LPS stimulation. As expected, the LPS-induced production of TNF-α, IL-6 and IL-1β were all signi?cantly increased both at mRNA and protein level. In conclusion, our data demonstrated that miR-709 could act as a negative regulator of macrophage in?ammatory responses by sup- pressing the production of pro-in?ammatory cytokines such as TNF-α, IL-6 and IL-1β. 3.3. MiR-709 regulates GSK-3β by directly targeting its 3′UTR To further study the molecular mechanisms underlying its anti- in?ammatory function, we performed computational analysis to identi- fy potential target genes of miR-709. Using online tools, putative seed match of miR-709 was predicted within the 3′UTR region of GSK-3β (Fig. 3A). To experimentally determine the interaction between miR-