Down-regulation of MicroRNA-34a* in Rheumatoid Arthritis Synovial Fibroblasts Promotes Apoptosis Resistance

Objective: To investigate the expression and effect of the microRNA-34 (miR-34) family on apoptosis in rheumatoid arthritis synovial fibroblasts (RASFs).

Methods: Expression of the miR-34 family in synovial fibroblasts with or without stimulation with Toll-like receptor (TLR) ligands, tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), hypoxia, or 5-azacytidine was analyzed by real-time polymerase chain reaction (PCR). Promoter methylation was studied by combined bisulfite restriction analysis. The effects of overexpression and silencing of miR-34a and miR-34a* on apoptosis were analyzed by annexin V/propidium iodide staining. Production of X-linked inhibitor of apoptosis protein (XIAP) was assessed by real-time PCR and immunohistochemistry analysis. Reporter gene assay was used to study the signaling pathways of miR-34a*.

Results: Basal expression levels of miR-34a* were found to be reduced in synovial fibroblasts from RA patients compared to osteoarthritis patients, whereas levels of miR-34a, miR-34b/b*, and miR-34c/c* did not differ. Neither TNFα, IL-1β, TLR ligands, nor hypoxia altered miR-34a* expression. However, we demonstrated that the promoter of miR-34a/34a* was methylated and showed that transcription of the miR-34a duplex was induced upon treatment with demethylating agents. Enforced expression of miR-34a* led to an increased rate of FasL- and TRAIL-mediated apoptosis in RASFs. Moreover, levels of miR-34a* were highly correlated with expression of XIAP, which was found to be up-regulated in RA synovial cells. Finally, we identified XIAP as a direct target of miR-34a*.

Conclusion: Our data provide evidence of a methylation-specific down-regulation of proapoptotic miR-34a* in RASFs. Decreased expression of miR- 34a* results in up-regulation of its direct target XIAP, thereby contributing to resistance of RASFs to apoptosis.