Crucial role of miR-9 and miR-155 in cartilage homeostasis and osteoarthritis pathology

Abstract

Several studies have reported beneficial effects of autophagy in preventing chondrocyte death, OA-like changes in gene expression and cartilage degeneration. Many microRNAs have been identified as key modulators of autophagy pathway. So far, to our knowledge no relationship has been revealed between nutraceuticals and microRNA network in OA. First aim of this thesis is to evaluate molecular mechanisms of action of hydroxytyrosol (HT) a promising compound already tested for protective efficacy in OA chondrocytes. HT increases markers of autophagy and protects chondrocytes from DNA damage and cell death induced by oxidative stress. The protective effect requires SIRT-1, which accumulated in the nucleus following HT treatment. Second aim consists in identifying a microRNA implicated in HT-mediated protective response to oxidative stress and examining the effects after modulation of miR levels by approach of transient transfection. After in silico analysis we identify miR-9 as a speculative candidate able to target SIRT-1 and confirm this hypothesis by means of luciferase gene-reporter assay. Moreover miR-9 mediates cell death induced by H2O2 and the protective effect of HT, as observed in human primary chondrocytes and C/28-I2 cell line. Third aim is to investigate the potential role of miR-155, found to be one of the most highly upregulated microRNAs in human OA knee cartilage, in autophagic pathway. Autophagy flux induced by rapamycin and 2-DG was significantly increased by miR-155 LNA, and significantly decreased after miR-155 mimic transfection in T/C28a2 cells and in human primary chondrocytes. These effects of miR-155 on autophagy were related to suppression of gene and protein expression of key autophagy. The identification of a single microRNA, involved simultaneously in several disease-related pathways, discloses a potent therapeutic target. Indeed the unveiling of bioactive compounds, exerting a beneficial effect through induction of epigenetic changes, may open a new topic of research not yet well explored.