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MiR-30a-3p negatively regulates BAFF synthesis in systemic sclerosis and rheumatoid arthritis fibroblasts.

Alsaleh G, François A, Philippe L, Gong YZ, Bahram S, Cetin S, Pfeffer S, Gottenberg JE, Wachsmann D, Georgel P, Sibilia J - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Immunorhumatologie moléculaire, INSERM UMR S_1109, Centre de Recherche en Immunologie et Hématologie, Faculté de Médecine, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France and Service de Rhumatologie, Centre National de Référence pour les Maladies Systémiques Autoimmunes Rares, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.

ABSTRACT
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.

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miR-30a-3p transfection affects BAFF mRNA expression and BAFF secretion in RAFLS and SScHDF.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.
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pone-0111266-g004: miR-30a-3p transfection affects BAFF mRNA expression and BAFF secretion in RAFLS and SScHDF.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.

Mentions: To assess the effect of miR-30a-3p on BAFF expression, we measured the production of BAFF mRNA by RT-qPCR in RAFLS and SScHDF transfected with miR-30a-3p mimic or with the AllStars negative siRNA control (CT). 24 h after transfection, cells were stimulated with Poly (I:C) or IFN-γ for 72 h. As seen in Figure 4, we found that overexpression of miR-30a-3p led to a global decrease in BAFF mRNA production and protein secretion by Poly (I:C)- and IFN-γ-activated RAFLS and SScHDF (panels A–D). Of note, transfection of miR-30a-3p mimic did not modulate IL-6 secretion by RAFLS and SScHDF activated with Poly (I:C) or IFN-γ (figure 4E–F), which is another major cytokine involved in B cells proliferation [23]. This indicates that miR-30a-3p does not interact with transcripts encoding factors involved in cytokine expression or inflammatory responses, but rather specifically interferes with BAFF mRNA in these cells, hence strengthening a potential role for BAFF in this process. Similar results were obtained upon transfection of miR-30e-3p and miR-30d-3p (data not shown).


MiR-30a-3p negatively regulates BAFF synthesis in systemic sclerosis and rheumatoid arthritis fibroblasts.

Alsaleh G, François A, Philippe L, Gong YZ, Bahram S, Cetin S, Pfeffer S, Gottenberg JE, Wachsmann D, Georgel P, Sibilia J - PLoS ONE (2014)

miR-30a-3p transfection affects BAFF mRNA expression and BAFF secretion in RAFLS and SScHDF.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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4216016&req=5

pone-0111266-g004: miR-30a-3p transfection affects BAFF mRNA expression and BAFF secretion in RAFLS and SScHDF.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.
Mentions: To assess the effect of miR-30a-3p on BAFF expression, we measured the production of BAFF mRNA by RT-qPCR in RAFLS and SScHDF transfected with miR-30a-3p mimic or with the AllStars negative siRNA control (CT). 24 h after transfection, cells were stimulated with Poly (I:C) or IFN-γ for 72 h. As seen in Figure 4, we found that overexpression of miR-30a-3p led to a global decrease in BAFF mRNA production and protein secretion by Poly (I:C)- and IFN-γ-activated RAFLS and SScHDF (panels A–D). Of note, transfection of miR-30a-3p mimic did not modulate IL-6 secretion by RAFLS and SScHDF activated with Poly (I:C) or IFN-γ (figure 4E–F), which is another major cytokine involved in B cells proliferation [23]. This indicates that miR-30a-3p does not interact with transcripts encoding factors involved in cytokine expression or inflammatory responses, but rather specifically interferes with BAFF mRNA in these cells, hence strengthening a potential role for BAFF in this process. Similar results were obtained upon transfection of miR-30e-3p and miR-30d-3p (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Immunorhumatologie moléculaire, INSERM UMR S_1109, Centre de Recherche en Immunologie et Hématologie, Faculté de Médecine, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France and Service de Rhumatologie, Centre National de Référence pour les Maladies Systémiques Autoimmunes Rares, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus