Purpose: Synovitis is a characteristic feature of both early and end-stage OA. However, the cellular mechanisms that regulate synovial tissue inflammation are poorly understood. Importantly, we recently identified through Next Generation Sequencing (NGS) several long non coding RNAs (lncRNAs) that were associated with the IL-1β inflammatory response in OA chondrocytes, and which regulated the secretion of pro-inflammatory cytokines including IL-6. The purpose of the present study was firstly to identify lncRNAs expressed in fibroblast-like synoviocytes (FLS) that were associated with inflammatory OA synovial tissue. Secondly to examine their expression in OA synovium compared to non-OA synovial tissue, and thirdly to determine whether modulation of the expression of an inflammatory-associated lncRNA affected the phenotype of isolated OA FLS cells.
Methods: Synovial tissue was collected from end-stage hip OA patients and from neck of femur fracture patients without OA (NOF#) undergoing total hip arthroplasty (NRES 14/ES/1044). Fibroblast-like synoviocytes (FLS) were cultured from synovial tissues and the culture media collected for the determination of IL-6 secretion by ELISA. Total RNA was extracted using Trizol (Invitrogen). RNA extracted from inflammatory and non-inflammatory FLS cells (n=4) was Ribozero treated, and subjected to RNA-seq (100bp paired-end, stranded sequencing) performed on the Illumina HiSeq-2000 sequencer. Differentially expressed lncRNAs were identified using the Galaxy Bioinformatics suite. Targeted knockdown of specific lncRNAs in FLS cells was performed using locked nucleic acids (LNAs; Exiqon) which were transfected by electroporation (AMAXA 4D). The effect of lncRNA knockdown on FLS cells was determined by assessment of the FLS phenotype by qRT-PCR, assessment of cytokine secretion (ELISA) and assessment of cell proliferation.
Results: 89 long intergenic non-coding RNAs (lincRNAs) were found to be differentially expressed (>1.5 fold, P<0.05) in FLS cells that were identified as inflammatory based on high IL-6 secretion. Analysis of 4 of these lncRNAs found that they also exhibited greater expression in OA synovium tissue, compared to non-OA NOF# synovium. Knockdown in the expression of one of the inflammatory lncRNAs in OA FLS cells using targeted LNA inhibitors (Exiqon) significantly reduced both the expression and secretion of IL-8 and reduced FLS cellular proliferation up to 5 days, compared to FLS cells transfected with a non-targeting control (NTC) LNA.
Conclusions: We have identified several lncRNAs that are differentially expressed in inflammatory OA FLS cells and which demonstrate differential expression between non-OA and OA synovium. Knockdown of an inflammatory-associated lncRNA in OA FLS cells reduced the expression and secretion of IL-8 and reduced FLS cellular proliferation. Further studies to determine both the function and mechanism of action of these inflammatory-associated lncRNAs in OA FLS cells, as well as their expression in other cell types of the synovium are warranted.
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