Identification of microRNA-181A-5P and microRNA-4454 as mediators of facet cartilage degeneration through the activation of zinc finger protein 440 mediated NF-KB signaling pathway and potential circulating biomarkers for detecting the severity of facet joint osteoarthritis in blood

Abstract

Purpose: Osteoarthritis (OA) of the spine (facet joints, FJ) is one of the major causes of severe low back pain and disability worldwide. However, specific mechanisms associated with facet cartilage degeneration during FJ OA are largely unknown. For the first time, in this study we aimed to investigate the role of microRNAs (miRNAs) in the pathophysiology of facet cartilage degeneration during FJ OA and the potential as biomarkers for detecting and determining the severity of FJ OA in human blood plasma. Methods: Based on MRI grading (by Weishaupt et al) and histopathology, we first established and validated a patient cohort (n = 203) (Group 1 patients [control group]: normal or mild facet cartilage degeneration and Group 2 patients [FJ OA group]: moderate to severe facet cartilage degeneration). Using this large and unique cohort, we screened 2,100 miRNAs using miRNA-array and differentially regulated the expressions of miRNAs in facet cartilage. miRNAs were further tested by qPCR analysis and their correlation with FJ OA severities were evaluated based on MRI grading score (grade 0; normal, grade 1; mild, grade 2; moderate and grade 3; severe). Human FJ OA chondrocytes were cultured and transfected with miRNA enhancers/inhibitors (or control enhancer/inhibitor) with/without interleukin (IL)-1β to determine the effect of miRNA enhancement/inhibition on the expression of catabolic (MMP13)/inflammatory (IL6, MCP1, TNFA)/anabolic (COL2A1)/cell death (PARP p85) markers by qPCR and western blot analysis. Target genes and signaling pathways modulated by miRNAs were also identified using mirDIP/pathDIP. Furthermore, we injected the miR-181a-5p in-vivo mimic (enhancer) into FJs of rats to see the effect of the overexpression in animal FJ cartilage. Finally, we tested the expression of two miRNAs in human blood plasma obtained from patients with various FJ OA severities (from grade 0 to grade 3) using qPCR analysis to identify any correlations between the circulating levels of two miRNAs in human blood plasma and FJ OA severity based on MRI grading. Results: Out of 2,100 miRNAs, we specifically identified 2 miRNAs (miR-181a-5p and miR-4454) whose expression were markedly up-regulated in FJ OA cartilage compared to control facet cartilage. Our data from patients with varying degrees of FJ OA disease severity showed that the expression levels of miR-181a-5p and miR-4454 in human facet cartilage were markedly elevated with increased degeneration and exhibited significant correlation with clinical disease severity based on MRI grading. We then treated FJ OA chondrocytes with miR-181a-5p or miR-4454 enhancer/inhibitor and showed that treatment with miR-181a-5p or miR-4454 enhancer significantly elevated the expression of inflammatory (IL6, MCP1, TNFA)/catabolic (MMP13)/cell death (PARP p85) markers and reduced expression of type II collagen (COL2A1). Inhibition of these two miRNAs was able to reverse these destructive effects compared to control enhancer/inhibitor in facet chondrocytes treated with IL-1β. Our study using mirDIP/pathDIP also identified that in facet cartilage both miR-181a-5p and miR-4454 signal zinc finger protein 440 (ZNF440) via modulation of the NF-κB pathway. Induction of NF-κB signaling using IL-1β treatment elevated miR-181a-5p and miR-4454 expression, which mediated a feedback loop to sustain NF-κB signaling through ZNF440 expression. FJ OA chondrocytes co-transfected with miR-181a-5p or miR-4454 mimic (or control mimic) and ZNF440 siRNA (or control siRNA) regulated phosphorylation of Ser536 NF-κB-p65 and reduction of IκB expression compared to control. Furthermore, by injecting miR-181a-5p in vivo-mimic (enhancer) in rat FJs, we observed a FJ OA phenotype in facet cartilage associated with enhanced catabolic activity and chondrocyte apoptosis in vivo. Finally, we determined that circulating forms of miR-181a-5p and miR-4454 in human blood plasma were sufficiently detectable and elevated in patient blood plasma with increased degree of FJ OA severity based on MRI grading. Conclusions: Using clinical, in vitro functional and in vivo studies, we have uniquely identified miR-181a-5p and miR-4454 as active players of facet cartilage degeneration during FJ OA through the activation of ZNF440-mediated NF-κB signaling pathway and potential circulating biomarkers in human blood plasma for detecting and determining the severity of FJ OA.