Long non-coding RNA expression profiling of subchondral bone reveals AC005165.1 modifying FRZB expression during osteoarthritis

Abstract

Objective: To gain insight in the expression profile of long non-coding RNAs (lncRNAs) in OA subchondral bone.

Methods: RNA sequencing data of macroscopically preserved and lesioned OA subchondral bone of patients that underwent joint replacement surgery due to OA (N = 22 pairs; 5 hips, 17 knees, Research osteoArthrits Articular Tissue (RAAK study) was run through an in-house pipeline to detect expression of lncRNAs. Differential expression analysis between preserved and lesioned bone was performed. Spearman correlations were calculated between differentially expressed lncRNAs and differentially expressed mRNAs identified previously in the same samples. Primary osteogenic cells were transfected with locked nucleic acid (LNA) GapmeRs targeting AC005165.1 lncRNA, to functionally investigate its potential mRNA targets.

Results: In total, 2816 lncRNAs were well-expressed in subchondral bone and we identified 233 lncRNAs exclusively expressed in knee and 307 lncRNAs exclusively in hip. Differential expression analysis, using all samples (N = 22 pairs; 5 hips, 17 knees), resulted in 21 differentially expressed lncRNAs [false discovery rate (FDR) < 0.05, fold change (FC) range 1.19-7.39], including long intergenic non-protein coding RNA (LINC) 1411 (LINC01411, FC = 7.39, FDR = 2.20 × 10-8), AC005165.1 (FC = 0.44, FDR = 2.37 × 10-6) and empty spiracles homeobox 2 opposite strand RNA (EMX2OS, FC = 0.41, FDR = 7.64 × 10-3). Among the differentially expressed lncRNAs, five were also differentially expressed in articular cartilage, including AC005165.1, showing similar direction of effect. Downregulation of AC005165.1 in primary osteogenic cells resulted in consistent downregulation of highly correlated frizzled related protein (FRZB).

Conclusion: The current study identified a novel lncRNA, AC005165.1, being dysregulated in OA articular cartilage and subchondral bone. Downregulation of AC005165.1 caused a decreased expression of OA risk gene FRZB, an important member of the wnt pathway, suggesting that AC005165.1 could be an attractive potential therapeutic target with effects in articular cartilage and subchondral bone.

Keywords: OA; articular cartilage; long non-coding RNA; subchondral bone.

Fig. 1

Schematic overview of applied strategy (A) Identification of expressed lncRNAs. (B) identification of lncRNAs differentially expressed between macroscopically preserved and lesioned OA subchondral bone. DE: differentially expressed; lncRNA: long non-coding RNA.

Fig. 2

Venn diagram Venn diagram of lncRNAs being expressed in the total, knee, and hip dataset of preserved and lesioned OA subchondral bone. lncRNA: long non-coding RNA.

Fig. 3

Heatmap of sample distance Heatmap is based on lncRNA expression levels of lncRNAs (N = 2057) expressed in all three datasets (i.e. total, hip and knee dataset of preserved and lesioned OA subchondral bone). lncRNA: long non-coding RNA.

Fig. 4

Volcano plot Volcano plot of differentially expressed lncRNAs in OA subchondral bone. The dots in the figure represent lncRNAs expressed in bone. Blue dots represent lncRNAs that are significantly differentially expressed, red dots represent lncRNAs that are significantly differentially expressed and have an absolute fold change of ≥2 and green dots represent the lncRNAs with an absolute fold change of ≥2 that are not significantly differentially expressed. FDR: false discovery rate; lncRNA: long non-coding RNA.

Fig. 5

Expression levels of AC005165.1, FRZB, CRIM1 and LVRN Expression levels of AC005165.1, FRZB, CRIM1 and LVRN upon either transfecting primary osteogenic cells with LNA GapmeRs targeting AC005165.1 (indicated with AC005165.1) or transfecting primary osteogenic cells with a negative control (cells were collected from N = 4 knee joints).