Introduction

MicroRNAs (miRNAs) are short non-coding RNAs that can regulate gene expression through base-specific interactions. They are generally found in intergenic regions carrying their own promoters or in intronic regions sharing a promoter with its host gene. They are first transcribed as primary miRNAs (pri-miRNA) with stem-loop structures. Still inside the nucleus they are processed into precursor (pre)-miRNAs and subsequently they are transported into the cytosol by Exportin-5. There, Dicer excises the miRNAs from the pre-miRNA hairpin stem and either alone or with Drosha, the strands of the double-stranded miRNA / miRNA* duplex are cleaved into single strands miRNAs. The single stranded miRNA can bind to the 3’ UTR region of a mRNA causing gene silencing. MiRNAs are involved in various cell processes and a dysregulation of miRNA expression is associated with a variety of pathologies.

Content

Dysregulated miRNA expression is associated with multiple cartilage pathologies, including rheumatoid arthritis and osteoarthritis (OA). In addition, it has been shown that modulation of a specific miRNA in chondrocytes can have an enormous effect on its metabolism and cartilage ECM homeostasis.

To identify miRNAs differentially expressed between OA and normal cartilage, total RNA was isolated from primary chondrons obtained from articular cartilage of normal knee joints post-mortem and of cartilage obtained from patients undergoing total knee arthroplasty. MiRNA expression was measured using TaqMan low-density Arrays A and B v3.0 Card sets. Gene target prediction with MirSystem was performed for the top 20% miRNAs with a significantly different expression between healthy and OA chondrons. The predicted genes were mainly involved in affecting the nuclear memory (e.g., epigenetic regulation), cytoarchitecture (e.g., cytoskeleton), protein turn-over (e.g., ubiquitination) and cell signaling (TGF-beta). These findings reflect multiple OA-related mechanisms by which miRNAs control gene expression in cartilage.

In addition, a screen to modulate the expression of 11 specific miRNAs was performed by transfecting passage 2 OA chondrocytes with miRNA precursor mimics or inhibitors and the appropriate mock negative controls during seeding at high density (1.26*10⁶ cells per cm²) on collagen-coated culture inserts. After 2 weeks of culture, glycosaminoglycan (GAG) content was determined by DMMB. Modulating the expression of hsa-miR-1, 21, 31, 126, 139 had no effect on GAG content, while hsa-miR-7, 15b, 20a, 27b, 148a and 432 increased GAG content after 2 weeks of culture. Overexpression of hsa-miR-7 positively effects cartilage formation by OA chondrocytes via the EGFR pathway and prevents the up-regulation of markers for hypertrophic differentiation, whereas hsa-miR-148a directly targets genes involved in cartilage catabolism.

Moreover, a library of 2048 miRNA mimics (Dharmacon) was transfected in GMP-compliant human bone marrow MSCs (3 donors) and assessed for early chondrogenesis in a high-throughput system. Six miRNA mimics that stimulated early chondrogenesis were transfected in MSCs that were cultured in pellets for four weeks. Hereafter, the pellets were analyzed for their collagen and glycosaminoglycan (GAG) content by biochemical assays and histology. Real time PCR was used to measure expression levels of chondrogenic and hypertrophic genes. In addition, alkaline phosphatase (ALP) activity was measured and potential gene targets of the miRNAs were identified using mirsystem. 22 miRNA mimics stimulated early chondrogenic differentiation. MiRNA mimics for hsa-miR-15b-3p, 138-5p, 139-3p, 432-5p, 520a-3p, and 520h increased GAG and total collagen content and type II collagen deposition in pellet cultures. No differences were found in aggrecan gene expression, overexpression of miR-15b-3p, 139-3p, 432-5p and 520h increased type II collagen gene expression levels. Increased ALP activity and type X collagen gene expression was found for hsa-miR-139-3p and 520a-3p. The miRNAs that stimulate chondrogenesis had 12 gene targets in common, mainly genes involved in the regulation of transcription. Gene ontology analysis of targets also identified pathways involved in neurotrophin signaling, osteoblast differentiation and cell morphogenesis.