Abstract
BackgroundOsteoarthritis (OA) is the most prevalent type of arthritis, which commonly involves inflammation in the articular cartilage in OA pathogenesis. MicroRNAs (miRNAs) play essential roles in the regulation and pathophysiology of various diseases including OA. MiR-410-3p has been demonstrated to mediate inflammatory pathways, however, the regulatory functions of miR-410-3p in OA remain largely unknown.MethodsThe regulations of miR-410-3p were investigated in OA. Mouse primary chondrocytes and mouse in vivo models were used. The expression levels of miR-410-3p and HMGB1 were measured by qPCR. The transcription activity of NF-κB was assessed by luciferase reporter assay. MTT assay was performed to assess cellular proliferation. Cell apoptosis was evaluated with the Fluorescein Isothiocyanate (FITC) Annexin V assay. Expression levels of proteins were determined by Western blot.ResultsThe results demonstrated that miR-410-3p was markedly downregulated in articular cartilage tissues as well as in lipopolysaccharide (LPS)-treated chondrocytes in OA mice. In addition, upregulation of miR-410-3p markedly inhibited LPS-induced apoptosis of chondrocytes. The results also demonstrated that the high mobility group box 1 (HMGB1) was a target of miR-410-3p. LPS-induced upregulated expression of HMGB1 significantly suppressed expression of miR-410-3p. Furthermore, upregulation of miR-410-3p markedly inhibited HMGB1 expression, the nuclear factor (NF)-kB activity and pro-inflammatory cytokines production. Taken together, the results suggested that miR-410-3p targeted HMGB1 and modulated chondrocytes apoptosis and inflammation through the NF-κB signaling pathway.ConclusionsThese findings provide insights into the potential of miR-410-3p/ HMGB1 as therapeutic targets for OA treatment.
Highlights
Osteoarthritis (OA) is the most prevalent type of arthritis, which commonly involves inflammation in the articular cartilage in OA pathogenesis
The articular cartilage were subdivision as three parts: one was primary Chondrocytes/articular cartilages of the medial tibial plateau, which was used for qPCR; the second part was primary Chondrocytes/articular cartilages of the medial tibial plateau that was used for western blot; the third part was knee joints of the right hind limbs, articular cartilages of the medial tibial plateau and medial femoral condyle, which was used for histology
Identification and verification of candidate HMGB1associated miRNAs Three miRNAs, miR-410-3p, miR-505-3p and miR129-5p, were considered as candidate miRNAs that were associated with high mobility group box 1 (HMGB1) based on ‘online available bio-informatic tools, including PTA, miRmap, microT, miRanda and targetScan (Fig. 1a)
Summary
Experimental animals and creation of OA models C57BL/6 background male mice (of Jackson origin) aged 8 weeks and weighed 20–25 g were purchased from the Animal Experimental Center of Affiliated Anqing Hospital of Anhui Medical University and were kept at a specific laboratory animal facility in pathogen-free microisolator cages with 12 h/12 h of light/dark at the temperature of 22–24 °C with free access to food and water. Eight weeks after the surgery, mice were euthanized by cervical dislocation and articular cartilage and synovial fluid were obtained by inserting a needle into the space between bones at the joint. Cell culture of mice chondrocytes Mouse primary articular chondrocytes were separated from tissues at knee joint as previously described [28]. Male mice aged 8 weeks were euthanized by cervical dislocation, and the articular cartilage tissue was carefully removed from the tibial plateau, femoral heads, and femoral condyles, following by rinsing with phosphate buffered saline (PBS). Elisa Protein levels in culturing supernatant and synovial fluid were determined by the Enzyme-Linked Immunosorbent Assay (ELISA) kit (R&D Systems) following the manufacturer’s protocol. Western blot Total proteins of cells and mouse articular cartilage tissues were isolated with RIPA buffer containing 1% protease inhibitors (Sigma-Aldrich). Significant difference was considered when P < 0.05
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