Background/Purpose:

In rheumatoid arthritis (RA) resident cells of the joint, fibroblast-like synoviocytes (RA FLS) acquire an aggressive phenotype in response to extrinsic factors such as PAMPs or DAMPs and intrinsic factors such as microRNAs. They produce large amounts of cytokines and among them the B cell-activating factor (BAFF) which allows them to collaborate with auto-immune B cells. We found that the miR-30* family of microRNAs (miR-30a*, d* and e*), which differ only 1 to 3 nucleotides, was predicted to potentially target the 3’-UTR region of BAFF. As BAFF is also up-regulated in the skin and the serum of systemic sclerosis patients, the aim of this study was to evaluate the role of miR-30* in the regulation of BAFF synthesis in fibroblasts isolated from either RA or SSc patients. 

Methods:

FLS and HDF were isolated from RA synovial tissues (n=6) or from skin from normal individual (n=3) or SSc patients (n=3) and were stimulated with TLR3 ligand (poly I:C) and IFN-γ for 3 days. RT-qPCR was performed to evaluate miRNA and mRNA expression. Transient transfection of RA-FLS and SSc-HDF with mimic miR-30* was performed using the Human Dermal Fibroblast NucleofectorTM kit from Amaxa. All assays were performed 24h post transfection. 

Results:

We first showed by qRT-PCR that like RA-FLS, SSc-HDF synthesized and released BAFF in response to poly I:C and IFN-γ. Conversely, HDF from normal subjects (NHDF) released BAFF only in response to IFN-γ. Using qRT-PCR, we demonstrated that miR-30a* and miR-30e* expression was strongly down regulated in RA-FLS, SScHDF and NHDF stimulated with either poly I:C or IFN-γ. Interestingly, NHDF which did not release BAFF in response to poly I:C expressed higher levels of miR-30a* and miR-30e* in response to poly I:C. MiR-30d* was not expressed constitutively or after activation by poly I:C and IFN-γ by each cell type. To evaluate whether miR-30* regulates BAFF expression in poly I:C and IFN-γ-activated RA-FLS and HDF, we transfected cells with miR-30* mimics. Transfection of the mimics induced a strong down-regulation of BAFF synthesis and release in response to poly I:C and IFN-γ. Moreover, we transiently transfected into HEK-293 cells a reporter construct that contain the firefly luciferase gene fused to the BAFF 3’-UTR containing the putative miR-30* interactor site along with miR-30*. We observed a downregulation of the luciferase activity, indicating that the 3’-UTR of BAFF mRNA is directly targeted by miR-30*.

Conclusion:

Our data strongly suggest a critical role of miR-30* in the regulation of the expression of BAFF which could play an important role in the regulation of the auto-immune response in RA and SSc.