The effect and mechanism of miRNA-140 in exosomes of hypoxic bone marrow mesenchymal stem cells on articular chondrocytes

Abstract

This study aimed to investigate the effects and mechanisms of miRNA-140 in exosomes of hypoxic bone marrow mesenchymal stem cells on articular chondrocytes. Articular chondrocytes were stimulated with IL-1β (10 μg/ml) to give them an inflammatory state. Exosomes were extracted by differential ultra-high speed centrifugation, and their morphology was identified by transmission electron microscopy. The Cell Counting Kit-8 (CCK-8) assay, cell scratch assay and flow cytometry method were utilized to assess the proliferation, migration and apoptosis of chondrocytes respectively. Expressions of miR-140, HIF-1α mRNA and mRNA associated with chondrocytes were investigated using quantitative reverse transcription PCR (qRT-PCR). Western blot was applied to assess the chondrocyte associated proteins Caspase3, Collagen II, HIF-1α, SOX-9 and BMSC-Exo surface protein markers CD9, CD63, TSG101. To observe effects of inflammatory chondrocytes, immunohistochemistry was adopted to detect the staining of Collagen I and Collagen II. Eventually, exosomes’ shape was almost round, and the scratch healing was significantly increased in BMSC-Exo treated groups compared with the IL-1β group (P < 0.001). Additionally, it was found that exosomes in the hypoxic state (Hypoxia-Exo) resulted in higher cellular activity, less apoptosis and enhanced protein expression in inflammatory chondrocytes compared to the normoxic state (Normoxia-Exo), while weakening adipose differentiation and enhancing chondrogenic differentiation. Furthermore, the healing effect of exosomes on inflammatory chondrocytes under hypoxic conditions was produced by a rise in miR-140 expression within them. In conclusion, under hypoxia, miRNA-140 in bone marrow mesenchymal stem cell exosomes promotes proliferation and migration of chondrocytes and reduces apoptosis of chondrocytes.