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

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

<p>See text for details.</p

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

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 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

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

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

MiR-30a-3p Negatively Regulates BAFF Synthesis in Systemic Sclerosis and Rheumatoid Arthritis Fibroblasts

<div><p>We evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.</p></div

miR-30a-3p expression in RAFLS and SScHDF regulates BAFF-dependent B cells survival in vitro.

<p>RAFLS (n = 4) (left) and SScHDF (n = 4) (right) were transfected with miR-30a-3p mimic (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with IFN-γ (0.1 or 5 ng/mL depending on the cell type) or medium for 72 h. Then, supernatants were harvested and cultured with purified blood B cells isolated from healthy subjects. B cells viability was determined by FACS analysis; vital B cells were brightly positive when stained with DiOC6 and excluded PI. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05.</p