Decreased expression of alpha-enolase inhibits the proliferation of hypoxia-induced rheumatoid arthritis fibroblasts-like synoviocytes

Abstract

Objectives. To investigate the effect of decreased alpha-enolase (ENO1) expression on rheumatoid arthritis fibroblasts-like synoviocytes (RA-FLSs) proliferation in response to hypoxia, and elucidate the possible mechanisms involved.

Methods. RA-FLSs and osteoarthritis fibroblasts-like synoviocytes (OA-FLSs) were cultured in tri-gas incubators with different oxygen concentrations (3% O₂, 7% O₂, and 21% O₂). 3% O₂ (hypoxia) and 7% O₂ conditions simulated intra-articular oxygen concentrations as observed in RA and healthy individual, respectively. 21% O₂ represented oxygen condition for normal cell culture. ENO1-knockdown FLSs were established using ENO1-siRNA. The expression level of ENO1 was detected using reverse transcription polymerase chain reaction or RT-PCR and Western blot. Proliferation and apoptosis of RA-FLSs and OA-FLSs were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium or MTS assay and flow cytometry, respectively. Western blot analysis was used to detect key proteins involved in apoptosis.

Results. ENO1 gene expression was remarkably upregulated, as well as its translation into protein, in RA-FLSs and OA-FLSs that were cultured in 3% O₂ concentration. RA-FLSs and OA-FLSs that were cultured under hypoxic conditions hyperproliferated compared with similar cells under normaxic conditions. Neither 7% O₂ nor 21% O₂ condition had any significant effect on ENO1 expression. ENO1-siRNA-transfected FLSs, but not control-siRNA FLSs, showed markedly decreased proliferation. Additionally, ENO1 expression was found to promote significantly higher expression levels of the anti-apoptotic proteins Bcl-2, surviving, and cyclinB1, but inhibited the expression of cleaved caspase3.

Conclusion. ENO1 may be crucial in the regulation of the proliferation and survival of synovial fibroblasts.

Keywords:

• Alpha-enolase
• Fibroblasts-like synoviocytes
• Hypoxia
• Proliferation
• Rheumatoid arthritis

Acknowledgements

The study was supported by grants from the National Natural Science Foundation of China (No.81373203).

Conflict of interest

None.