Abstract
OBJECTIVE: We investigated the expression and the impact of the microRNA-34 (miR-34) family on apoptosis in synovial fibroblasts (SF) of rheumatoid arthritis (RA) patients. METHODS: Expression of the miR-34 family was analyzed by real-time PCR in SF with or without stimulation with Toll-like receptor (TLR) ligands, TNF-α, IL-1β, hypoxia and 5-azacytidine. Promoter methylation was studied by combined bisulfite restriction analysis. Overexpression and silencing of miR-34a and miR-34a* was used to analyze their effect on apoptosis by annexin V/PI staining. Production of XIAP (X-linked inhibitor of apoptosis protein) was analyzed by real-time PCR and immunohistochemistry. Reporter gene assay was used to study the signaling pathways of miR-34a*. RESULTS: Basal expression levels of miR-34a*, but not of miR-34a, miR-34b/b* and miR-34c/c*, were found to be reduced in SF from RA compared to osteoarthritis. Neither TNF-α, IL-1β, TLR ligands nor hypoxia altered miR-34a* expression. However, we identified the promoter of miR-34a/34a* to be methylated and showed that transcription of the miR-34a duplex is induced upon treatment with demethylating agents. Enforced expression of miR-34a* led to an increased rate of FasL and TRAIL mediated apoptosis in RASF. Moreover, levels of miR-34a* highly correlated with the expression of XIAP, which was found to be upregulated in RA synovial cells. Finally, our study identified XIAP as a direct target of miR-34a*. CONCLUSION: Our data provide evidence for a methylation specific downregulation of pro-apoptotic miR-34a* in RASF. Decreased expression of miR-34a* results in upregulation of its direct target XIAP, thereby contributing to apoptosis resistance of RASF.
Niederer, F