Background/Purpose: Rheumatoid arthritis (RA) is characterized by inflammation and subsequent infiltration of peripheral blood monocytes (MNs) into the synovial tissue (ST). Micro (mi)RNAs are non-coding RNAs which regulate many physiological and pathological functions at the posttranscriptional level. We performed miRNA PCR array and found a panel of miRNAs, including miR429, were highly expressed in response to TNF-α in MNs. Hence we examined the contribution miR429 and one of its target genes, PAK2, in MN chemotaxis and acute inflammatory arthritis.

Methods: The expression of miR429 in RA and NL MNs was assessed via quantitative (q)PCR. RA MNs and RA fibroblast-like synoviocytes (FLS) were transfected with inhibitors and mimics of miR429 for 24 hours. Then these cells were stimulated with TNF-α for 25 minutes to determine phosphorylation of signaling molecules by Western blotting. We transfected U937 cells, a human leukemic MN lymphoma cell line, and NL MNs with miR429 mimics and inhibitors and performed chemotaxis. To investigate the role of miR429 in acute inflammation, miR429 shRNA or control shRNA were packaged into lentivirus and injected into mouse knees 30 minutes prior to administration of TNF-α or Zymosan. Mouse knee circumference was measured before injections and again 24 hours later. Following euthanasia, knees were harvested for immunofluorescence staining or homogenized for analysis of inflammatory cytokines.

Results: Non-stimulated RA MNs had a 2.6 fold increase in miR429 expression compared to non-stimulated Normal (NL) MNs by qPCR. We also found that miR429 is inducible, TNF-α induced significantly higher miR429 expression in NL MNs compared to non-stimulated MNs. With regards to MN migration, inhibition of miR429 decreased migration of U937 cells and MNs in response to MCP1/CCL2, suggesting a role for miR429 in MN migration. TNF-α upregulation of phospho-Erk, -JNK and -NFkB was decreased when RA MNs were transfected with miR429 inhibitor. Similarly, a decrease of TNF-α induced phospho-Erk, -JNK and -NFkB was observed in RA FLS. Furthermore we also determined that a miR429 target gene, PAK2, was downregulated when miR429 was inhibited in RA MNs and RA FLS. Additionally, TNF-α and Zymosan treated mouse knee swelling was significantly reduced in mice injected with miR429 inhibitor compared to control, indicating that miR429 is critical to arthritis development in vivo. We also found decreased ingress of leukocytes into mouse knee cryosections treated with miR429 inhibitor.

Conclusion: miR429 expression is increased with TNF-α stimulation and has higher levels in RA compared to NL MNs. miR429 plays an essential role in the migration of MNs and also regulates phosphorylation of signaling molecules and PAK2 transcription factor. Additionally mice treated with miR429 inhibitor showed decreased inflammation indicating the importance of miR429 in acute inflammation and MN recruitment. This data suggests that miR429 may be a therapeutic target in MN dependent diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/unique-role-for-mir429-in-ra-and-acute-model-of-arthritis/