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 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-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

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 transfection affects BAFF mRNA expression and BAFF secretion in RAFLS and SScHDF.

<p>A, C. RAFLS (n = 5) (A) and SScHDF (n = 4) (C) were transfected with miR-30a-3p mimic (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with Poly (I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL, depending on cell types) or medium for 72 h. BAFF mRNA expression was determined by RT-qPCR. Results were normalized to Gapdh and expressed as fold change compared with samples from cells incubated with medium. B, D. BAFF release was determined by ELISA in culture supernatants in the same conditions as panel A and C. E, F. IL-6 release was determined by ELISA in culture supernatants in the same conditions as panel A and C. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05; **p<0.01; ***p<0.001.</p