Effect of transfection of miR-346 antagomirs on TNF-α mRNA stability and release in RA FLS.

<p><b>A.</b> FLS were transfected with miR-346 antisense molecules or with the Clear-miR™ negative control (control), activated with LPS 24 h post-transfection for 3 h and incubated for another 1, 2, 3 and 4 h with actinomycin D. Control cells were incubated for 3 h with medium (C). NT: non transfected cells. <b>B, C</b>: TNF-α expression was detected using cellular ELISA or western blotting with anti-TNF-α antibodies, in FLS transfected with miR-346 antisense molecules or with the Clear-miR™ negative control (control) or in non transfected FLS (NT). 24 h post-transfection, FLS were either incubated in medium (C) or activated with LPS for 6 h. The results are representative of three different experiments for each patient. <b>D.</b> TNF-α release was determined by ELISA in culture supernatants after stimulation with LPS or medium (C). Data are expressed as the mean of triplicate samples +/− SD of three independent experiments for each patient. p<0.01.</p

Model of action for miR-346 and Btk in the regulation of TNF-α secretion.

<p>See text for details.</p

Effect of LPS on TNF-α release by RA FLS and THP-1 cells.

<p><b>A</b>. IL-6 release was determined by ELISA in culture supernatants harvested 3 h, 6 h and 24 h after stimulation with LPS (1 µg/ml) or medium (C). <b>B, C</b>. TNF-α release by RA FLS and THP-1 cells was determined by ELISA in culture supernatants harvested 3 h, 6 h and 24 h after stimulation with LPS or medium (C). Data are expressed as the mean of triplicate samples ± SD and are representative of three independent experiments. <b>D</b>. TNF-α expression was determined by western-blotting with anti-TNF-α antibodies in RA FLS, 3 h, 6 h and 24 h after stimulation with LPS or medium (C). Recombinant TNF-α was used as control. For protein loading control, membranes were reprobed with anti-β-actin antibodies. Data are expressed as the mean of triplicate samples +/− SD of three independent experiments for each patient.</p

Effect of LPS on TNF-αmRNA expression in RA FLS and THP-1 cells.

<p><b>A, B.</b> TNF-α mRNA expression was determined by RT-PCR in RA FLS (<b>A</b>) and THP-1 cells (<b>B</b>) stimulated with LPS (1 µg/ml) for 2 h, 4 h and 6 h. Control cells were incubated for 4 h with medium (C). <b>C, D.</b> RA FLS and THP-1 cells were stimulated for 3 h with LPS and then incubated for another 1, 2, 3 and 4 h with actinomycin D (5 µg/ml). Control cells were incubated for 3 h with medium. TNF-α mRNA expression was determined by RT-PCR. The results are representative of three different experiments for each patient.</p

Effect of transfection of miR-346 mimic on TNF-α release by THP-1 cells.

<p><b>A.</b> TNF-α release by THP-1 cells preincubated or not (control) with LFMA-13 (172 mM) for 1 h and then stimulated with LPS or medium (C) for 3 and 6 h, was evaluated by ELISA. <b>B.</b> THP-1 cells were transfected with miR-346 mimic or with the miRNA mimic negative control (control) and activated 24 h post-transfection with either LPS or medium (C) for 3 and 6 h. TNF-α release was evaluated by ELISA. Data are expressed as the mean of triplicate samples +/− SD of three independent experiments for each patient. p<0.01.</p

Quantitative RT-PCR analysis of miR-346 expression in LPS-activated RA FLS and THP-1 cells.

<p>MiR-346 level was determined by quantitative RT-PCR in RA FLS and THP-1 cells stimulated with LPS (1 µg/ml) for 3 h and 6 h. U6 small nuclear RNA (RNAU6) was used as endogenous control for data normalization. The control (C) corresponded to untreated cells. Data are expressed as the mean of triplicate samples +/− SD of three independent experiments for each patient.</p

Effect of transfection of miRNA antisense molecules on TNF-αsynthesis by RA FLS.

<p><b>A, B.</b> MiR-125b and miR-939 levels were determined by quantitative RT-PCR in RA FLS and THP-1 cells stimulated with LPS for 3 h and 6 h. RNAU6 was used as endogenous control for data normalization. The control (C) corresponded to untreated cells. RA FLS were transfected with either miR-125b or miR-939 antisense molecules or in combination or with the Clear-miR™ negative control (control). LPS or medium (C) activation of transfected cells was performed 24 h post-transfection, for 3 h and 6 h. Non transfected RA FLS were used as negative controls (NT). <b>C, D.</b> TNF-α and IL-6 release were determined by ELISA in culture supernatants harvested 6 h after stimulation with LPS or medium (C). <b>E.</b> Intracellular TNF-α expression was determined in transfected FLS and activated with LPS for 24 h. Data are expressed as the mean of triplicate samples +/− SD of three independent experiments for each patient.</p

MiR-346 regulates the expression,of TTP in activated RA FLS and THP-1 cells.

<p><b>A, B:</b> TTP mRNA levels were determined using quantitative RT-PCR in LPS-activated THP-1 cells transfected with miR-346 antisense molecules or preincubated for 1 h with LFMA-13. <b>C:</b> TTP mRNA levels were determined using quantitative RT-PCR in LPS-activated RA-FLS transfected with miR-346 antisense molecules Results were normalized to GAPDH and expressed as fold change compared with samples from cells incubated in medium (C). <b>D, E:</b> THP-1 cells were transfected with miR-346 mimic, with miR-346 mimic and siRNATTP or with a negative control (control) and activated 48 h post-transfection with either LPS or medium (C). TNF-α mRNA levels were determined using RT-PCR and TNF-α release was evaluated by ELISA. Data are expressed as the mean of triplicate samples +/− SD of three independent experiments for each patient.</p