MiR-1285-5p Targeting PAK1 Alleviates Knee OA by NF-κB Signaling Pathway in Vitro

Abstract

Background: An increasing number of microRNAs (miRNAs) are involved in the progression of knee osteoarthritis (OA). In this study, we identified a previously unknown role of miR-1285-5p in inflammation during knee OA and its underlying mechanism. Methods: RT-qPCR (real time flurocent qualitative PCR) was used to detect the expression of miR-1285-5p and PAK1 (p21 activated kinase 1) in cartilage from subjects with knee OA, normal tissues, and chondrocytes. After establishing in vitro chondrocyte inflammation model, we detected cell proliferation and apoptosis using CCK-8 (cell counting kit-8), EdU proliferation, flow cytometry, and caspase-3 activity assays. ELISA (enzyme-linked immunosorbent assay) was also used to test IL-1β (interleukin-1β), TNF-α (tumor necrosis factor alpha), and IL-6 levels in the chondrocyte culture. The targeted binding of miR-1285-5p to the PAK1 3′UTR (untranslational region) was verified using luciferase reporter assays. Results: MiR-1285-5p was downregulated in cartilage from subjects with knee OA and lipopolysaccharide (LPS)-treated chondrocytes. MiR-1285-5p overexpression inhibited the increased secretion of IL-1β, TNF-α, and IL-6 in chondrocytes after LPS treatment. Similarly, inhibition overexpression was observed on LPS-induced chondrocyte apoptosis after miR-1285-5p overexpression. In contrast, miR-1285-5p overexpression restored the proliferative impairment in chondrocytes exposed to LPS. MiR-1285-5p targeted and suppressed PAK1 expression. PAK1 overexpression abrogated the protective effect of the miR-1285-5p mimic on LPS-stimulated chondrocytes. The relieving effect of miR-1285-5p on LPS-increased p-IKKβ (phospho-IKK beta), IκB-α (IkappaB-alpha), and NF-κBp65 (nuclear factor NF-kappa-B p65) levels was offset by PAK1 overexpression. Conclusions: By downregulating PAK1 expression via NF-κB signaling, miR-1285-5p protection against knee OA was demonstrated, indicating that miR-1285-5p/PAK1 may be a suitable therapeutic target for the management of human knee OA.