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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 directly targets the 3′UTR of BAFF mRNA.A. Luciferase reporter constructs with wild-type or mutated (for miR-30-3p binding sites) BAFF 3′UTR were generated. B. HEK293 cells were transiently co-transfected with reporter constructs and with miR-30a-3p mimic (20 pM). Firefly Luciferase activities were measured 48 h after transfection and normalized to Renilla Luciferase expressed by the control psi-CHECK-2 vector devoid of 3′UTR sequences. Data are expressed as the mean of triplicate samples ± SEM and are representative of three independent experiments. **p<0.01.
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pone-0111266-g003: miR-30a-3p directly targets the 3′UTR of BAFF mRNA.A. Luciferase reporter constructs with wild-type or mutated (for miR-30-3p binding sites) BAFF 3′UTR were generated. B. HEK293 cells were transiently co-transfected with reporter constructs and with miR-30a-3p mimic (20 pM). Firefly Luciferase activities were measured 48 h after transfection and normalized to Renilla Luciferase expressed by the control psi-CHECK-2 vector devoid of 3′UTR sequences. Data are expressed as the mean of triplicate samples ± SEM and are representative of three independent experiments. **p<0.01.

Mentions: To validate the involvement of miR-30a-3p in the regulation of BAFF expression, we generated luciferase reporter constructs (derived from the pSI-CHECK2 vector) containing the firefly luciferase gene fused to the entire human BAFF 3′UTR sequence and the renilla luciferase for normalization. We also generated a reporter construct in which a mutated version, designed to disrupt the predicted seed-match for miR-30a-3p of the human BAFF 3′UTR, was inserted (figure 3A). These plasmids were co-transfected in HEK293 cells with miR-30a-3p mimic or AllStars negative control siRNA (CT). In the presence of miR-30a-3p mimic, we observed a significant down regulation of the BAFF 3′UTR-controlled luciferase sensor, whereas luciferase expression upon transfection of the mutated form of the reporter vector remained unchanged (figure 3B). Altogether, these data suggest that BAFF transcripts can be directly targeted by miR-30a-3p for post-transcriptional regulation.


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 directly targets the 3′UTR of BAFF mRNA.A. Luciferase reporter constructs with wild-type or mutated (for miR-30-3p binding sites) BAFF 3′UTR were generated. B. HEK293 cells were transiently co-transfected with reporter constructs and with miR-30a-3p mimic (20 pM). Firefly Luciferase activities were measured 48 h after transfection and normalized to Renilla Luciferase expressed by the control psi-CHECK-2 vector devoid of 3′UTR sequences. Data are expressed as the mean of triplicate samples ± SEM and are representative of three independent experiments. **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111266-g003: miR-30a-3p directly targets the 3′UTR of BAFF mRNA.A. Luciferase reporter constructs with wild-type or mutated (for miR-30-3p binding sites) BAFF 3′UTR were generated. B. HEK293 cells were transiently co-transfected with reporter constructs and with miR-30a-3p mimic (20 pM). Firefly Luciferase activities were measured 48 h after transfection and normalized to Renilla Luciferase expressed by the control psi-CHECK-2 vector devoid of 3′UTR sequences. Data are expressed as the mean of triplicate samples ± SEM and are representative of three independent experiments. **p<0.01.
Mentions: To validate the involvement of miR-30a-3p in the regulation of BAFF expression, we generated luciferase reporter constructs (derived from the pSI-CHECK2 vector) containing the firefly luciferase gene fused to the entire human BAFF 3′UTR sequence and the renilla luciferase for normalization. We also generated a reporter construct in which a mutated version, designed to disrupt the predicted seed-match for miR-30a-3p of the human BAFF 3′UTR, was inserted (figure 3A). These plasmids were co-transfected in HEK293 cells with miR-30a-3p mimic or AllStars negative control siRNA (CT). In the presence of miR-30a-3p mimic, we observed a significant down regulation of the BAFF 3′UTR-controlled luciferase sensor, whereas luciferase expression upon transfection of the mutated form of the reporter vector remained unchanged (figure 3B). Altogether, these data suggest that BAFF transcripts can be directly targeted by miR-30a-3p for post-transcriptional regulation.

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