Previous studies have reported miR-217 uregulation in age-related pathologies. We investigated the impact of miR-217-5p on sirtuin 1 (SIRT1) regulation in human osteoarthritic (OA) chondrocytes. MiR-217 target enrichment analyses were performed using three public databases, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. MiR-217-5p expression levels were quantified in normal and OA chondrocytes. SIRT1 expression levels, nuclear factor kappa-B p65 subunit (NF-κBp65) and p53 acetylation levels, and expression levels of OA-related pro-inflammatory markers [tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), IL-6], pro-apoptotic markers [Bax, pro-caspase 3, cleaved caspase 3] and matrix regulators [matrix metalloproteinase (MMP)-1, MMP-13, MMP-9, Collagen 2 (COL2A1), Aggrecan (ACAN)] were evaluated in miR-217 mimic-treated and/or miR-217 inhibitor-treated OA chondrocytes, with/without subsequent treatment with siRNA against SIRT1 (siSIRT1). MiR-217-5p was upregulated in OA chondrocytes, while target prediction/enrichment analyses revealed SIRT1 as miR-217 target-gene. Deacetylation of NF-κBp65 and p53 in miR-217 inhibitor-treated OA chondrocytes was reversed by siSIRT1 treatment. MiR-217 inhibitor-treated OA chondrocytes showed increased COL2A1, ACAN and decreased IL-1β, IL-6, TNFα, Bax, cleaved caspase 3 and MMPs expression levels, which were reversed following miR-217 inhibitor/siSIRT1 treatment. Our findings highlight the impact of miR-217-5p on SIRT1 downregulation contributing to OA pathogenesis.

Small extracellular vesicles (sEVs) derived from different osteoarthritic (OA) tissues regulate OA-related biological processes through transporting their content (proteins, miRNAs, etc.) to recipient cells. This study aimed to characterize the miRNA profile of synovial fibroblasts-derived small EVs (FS_OA_sEVs) and investigate their role in inflammation in chondrocytes. Chondrocytes were isolated from macroscopically preserved and lesioned OA cartilage (C_OAmin and C_OAmax) and synovial fibroblasts from OA synovium. Synovial fibroblasts-derived small EVs (FS_OA_sEVs) were characterized according to ISEV guidelines and used for miRNA profiling and bioinformatics analysis. miR-21-5p was identified as one of the most abundant, and its target genes, such as KLF6, were enriched in OA-related processes including inflammation. Treatment of C_OAmin chondrocytes with FS_OA_sEVs resulted in decreased expression of COL2A1 and ACAN and an increase in catabolic markers MMP-3 and MMP-13. Moreover, C-OAmin receiving FS_OA_sEVs exhibited increased levels of inflammatory markers and miR-21-5p expression, resembling chondrocytes’ phenotype from lesioned OA cartilage, whereas miR-21-5p inhibition reversed their expression of inflammatory markers and miR-21-5p. Compared to C_OA min, C_OAmax chondrocytes exhibited increased miR-21-5p and inflammatory markers expression and decreased KLF6 expression. miR-21-5p inhibition in C_OAmax led to KLF6 upregulation and suppression of inflammatory mediators, whereas co-treatment with siRNA against KLF6 negated this effect, confirming a potential direct regulatory relationship between miR-21-5p and KLF6. Our results provide novel insights into the FS_OA_sEV-mediated inflammation axis, highlighting FS_OA_sEV-derived miR-21-5p as a driver of OA progression via regulating inflammation in chondrocytes.

Although MSCs grant pronounced potential for cell therapies, several factors, such as their heterogeneity restrict their use. To overcome these limitations, iMSCs (MSCs derived from induced pluripotent stem cells (iPSCs) have attracted attention. Here, we analyzed the transcriptome of MSCs, iPSCs and iMSCs derived from healthy individuals and osteoarthritis (OA) patients and explored miRNA-mRNA interactions during these transitions. We performed RNA-seq and gene expression comparisons and Protein-Protein-Interaction analysis followed by GO enrichment and KEGG pathway analyses. MicroRNAs’ (miRNA) expression profile using miRarrays and differentially expressed miRNA’s impact on regulating iMSCs gene expression was also explored. Our analyses revealed that iMSCs derivation from iPSCs favors the expression of genes conferring high proliferation, differentiation, and migration properties, all of which contribute to a rejuvenated state of iMSCs compared to primary MSCs. Additionally, our exploration of the involvement of miRNAs in this rejuvenated iMSCs transcriptome concluded in twenty-six miRNAs that, as our analysis showed, are implicated in pluripotency. Notably, the identified here interactions between hsa-let7b/i, hsa-miR-221/222-3p, hsa-miR-302c, hsa-miR-181a, hsa-miR-331 with target genes HMGA2, IGF2BP3, STARD4, and APOL6 could prove to be the necessary tools that will convey iMSCs into the ideal mean for cell therapy in osteoarthritis.

Background: Pediatric thrombosis (PT) represents a rare condition that can manifest from neonatal life to adolescence, encompassing life-threatening complications. Its pathogenesis is attributed to immature hemostasis in conjunction with environmental and genetic factors, predominantly including those resulting in increased levels of plasminogen activator inhibitor 1 (PAI-1), the principal inhibitor of fibrinolysis, subject to upstream regulation by ACE (angiotensin-converting enzyme). Although the implication of microRNAs (miRNAs), epigenetic modulators of gene-expression, is demonstrated in adult-thrombosis, evidence is lacking in the pediatric setting. Here, we investigated the involvement of two miRNA-regulators of PAI-1 in PT, in relation to clinical and genetic parameters that induce PAI-1 fluctuations. Methods: Following bioinformatic target-prediction, miRNA expression was assessed by quantitative real-time PCR, in serum-samples of 19 pediatric patients with thrombosis (1-18 months post-incident), and 19 controls. Patients were genotyped for the PAI-1-4G/5G and ACE-I/D polymorphisms by PCR-based assays. Genotypic and thrombosis-related clinical data were analyzed in relation to miRNA-expression. Results: Two miRNAs (miR-145-5p, miR-34a-5p) were identified to target PAI-1 mRNA, with miR-34a additionally targeting ACE mRNA. The expression of miR-34a was significantly decreased in patients compared to controls (p=0.029), while no difference was observed for miR-145. Within patients, miR-34a demonstrated a peak 1-3 months post-thrombosis and was diminished upon treatment-completion (p=0.031), followed by a slight long-term increase. MiR-34a expression differed significantly by thrombosis site (p=0.019), while a significant synergistic interaction between site and onset-type (provoked/unprovoked) was detected (p=0.016). Finally, an epistatic modification was observed in cerebral cases, since double homozygosity (4G/4G+D/D) led to miR-34a diminishing, with DD carriership reversing the 4G4G-induced upregulation of miR-34a (p=0.006). Conclusions: Our findings suggest that in pediatric thrombosis, downregulation of miR-34a is indicative of impaired fibrinolytic capacity, attributed to deficient regulation of the inhibitory ACE/PAI-1 axis.