hsa-miR-15a exerts protective effects against osteoarthritis by targeting aggrecanase-2 (ADAMTS5) in human chondrocytes

摘要

The aim of the present study was to examine the expression levels and role of hsa-miR-15a in osteoarthritis (OA), as well as the associated mechanisms. The expression levels of hsa-miR-15a and A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (ADAMTS5, also known as aggrecanase-2) were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in both OA and normal chondrocytes. hsa-miR-21 mimics or antisense oligonucleotides (ASO) were co-transfected into the chondrocytes to examine the effects on the putative binding sites compared with the negative control (NC)-mimics or NC-ASO. The relative ADAMTS5 mRNA and protein levels were measured by RT-qPCR and western blot anlaysis, respectively. Moreover, after inhibiting the expression of hsa-miR-15a and ADAMTS5 by ASO and small interfering RNA (siRNA), respectively, the amounts of proteoglycan and collagen in the cellular matrix and medium were determined. Additionally, the expression levels of collagen II were measured by western blot analysis. hsa-miR-15a expression was downregulated, but ADAMTS5 expression was upregulated in the human OA chondrocytes compared to the normal chondrocytes. Luciferase reporter assay confirmed that the hsa-miR-15a binding site was in the ADAMTS5 gene 3'-untranslated region (3'-UTR), and ADAMTS5 was negatively regulated by hsa-miR-15a. The downregulation of hsa-miR-15a decreased the aggregation of proteoglycan and the collagen content, but increased the release of proteoglycan and collagen; total collagen production was significantly lower, and collagenase activity was markedly higher. The downregulation of ADAMTS5 increased the aggregation of proteoglycan and the collagen content, but decreased the release of proteoglycan and collagen, along with total collagen production. Moreover, collagenase activity was markedly lower. The findings of our study suggest that hsa-miR-15a exerts protective effects against OA by targeting ADAMTS5 in human chondrocytes.