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

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

Tuerlings M ，van Hoolwerff M ，van Bokkum JM ，Suchiman HED ，Lakenberg N ，Broekhuis D ，Nelissen RGHH ，Ramos YFM ，Mei H ，Cats D ，Coutinho de Almeida R ，Meulenbelt I ... -  《-》

RNA Sequencing Reveals Interacting Key Determinants of Osteoarthritis Acting in Subchondral Bone and Articular Cartilage: Identification of IL11 and CHADL as Attractive Treatment Targets.

To identify key determinants of the interactive pathophysiologic processes in subchondral bone and cartilage in osteoarthritis (OA). We performed RNA sequencing on macroscopically preserved and lesional OA subchondral bone from patients in the Research Arthritis and Articular Cartilage study who underwent joint replacement surgery due to OA (n = 24 sample pairs: 6 hips and 18 knees). Unsupervised hierarchical clustering and differential expression analyses were conducted. Results were combined with data on previously identified differentially expressed genes in cartilage (partly overlapping samples) as well as data on recently identified OA risk genes. We identified 1,569 genes that were significantly differentially expressed between lesional and preserved subchondral bone, including CNTNAP2 (fold change [FC] 2.4, false discovery rate [FDR] 3.36 × 10-5 ) and STMN2 (FC 9.6, FDR 2.36 × 10-3 ). Among these 1,569 genes, 305 were also differentially expressed, and with the same direction of effect, in cartilage, including the recently recognized OA susceptibility genes IL11 and CHADL. Upon differential expression analysis with stratification for joint site, we identified 509 genes that were exclusively differentially expressed in subchondral bone of the knee, including KLF11 and WNT4. These genes that were differentially expressed exclusively in the knee were enriched for involvement in epigenetic processes, characterized by, e.g., HIST1H3J and HIST1H3H. IL11 and CHADL were among the most consistently differentially expressed genes OA pathophysiology-related genes in both bone and cartilage. As these genes were recently also identified as robust OA risk genes, they classify as attractive therapeutic targets acting on 2 OA-relevant tissues.

Tuerlings M ，van Hoolwerff M ，Houtman E ，Suchiman EHED ，Lakenberg N ，Mei H ，van der Linden EHMJ ，Nelissen RRGHH ，Ramos YYFM ，Coutinho de Almeida R ，Meulenbelt I ... -  《-》

Elucidating Epigenetic Regulation by Identifying Functional cis-Acting Long Noncoding RNAs and Their Targets in Osteoarthritic Articular Cartilage.

To identify robustly differentially expressed long noncoding RNAs (lncRNAs) with osteoarthritis (OA) pathophysiology in cartilage and to explore potential target messenger RNA (mRNA) by establishing coexpression networks, followed by functional validation. RNA sequencing was performed on macroscopically lesioned and preserved OA cartilage from patients who underwent joint replacement surgery due to OA (n = 98). Differential expression analysis was performed on lncRNAs that were annotated in GENCODE and Ensembl databases. To identify potential interactions, correlations were calculated between the identified differentially expressed lncRNAs and the previously reported differentially expressed protein-coding genes in the same samples. Modulation of chondrocyte lncRNA expression was achieved using locked nucleic acid GapmeRs. By applying our in-house pipeline, we identified 5,053 lncRNAs that were robustly expressed, of which 191 were significantly differentially expressed (according to false discovery rate) between lesioned and preserved OA cartilage. Upon integrating mRNA sequencing data, we showed that intergenic and antisense differentially expressed lncRNAs demonstrate high, positive correlations with their respective flanking sense genes. To functionally validate this observation, we selected P3H2-AS1, which was down-regulated in primary chondrocytes, resulting in the down-regulation of P3H2 gene expression levels. As such, we can confirm that P3H2-AS1 regulates its sense gene P3H2. By applying an improved detection strategy, robustly differentially expressed lncRNAs in OA cartilage were detected. Integration of these lncRNAs with differential mRNA expression levels in the same samples provided insight into their regulatory networks. Our data indicates that intergenic and antisense lncRNAs play an important role in regulating the pathophysiology of OA.

van Hoolwerff M ，Metselaar PI ，Tuerlings M ，Suchiman HED ，Lakenberg N ，Ramos YFM ，Cats D ，Nelissen RGHH ，Broekhuis D ，Mei H ，de Almeida RC ，Meulenbelt I ... -  《-》

Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage.

To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA. OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non-OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase-polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex. RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin-1β (IL-1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50-associated cyclooxygenase 2-extragenic RNA (PACER) and 2 novel chondrocyte inflammation-associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non-OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL-1-stimulated secretion of proinflammatory cytokines. The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation-driven cartilage degeneration in OA.

Pearson MJ ，Philp AM ，Heward JA ，Roux BT ，Walsh DA ，Davis ET ，Lindsay MA ，Jones SW ... -  《-》

Expression profile of long noncoding RNAs in cartilage from knee osteoarthritis patients.

Long noncoding RNAs (lncRNAs) have emerged as a novel class of regulatory molecules involved in various biological processes, but their role in osteoarthritis (OA) remains unknown. Therefore, we aimed to reveal lncRNAs expression profile in human osteoarthritic cartilage and explore the potential functions of lncRNAs in OA. The expression profiles of lncRNAs and mRNAs in OA cartilage were obtained using microarray and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bioinformatics analyses including lncRNA classification and subgroup analysis, gene ontology (GO) analysis, pathway analysis, network analysis and target prediction were performed. There were 3007 upregulated lncRNAs and 1707 downregulated lncRNAs in OA cartilage compared with normal samples (Fold change ≥ 2.0). In addition, 2136 mRNAs were upregulated and 2,241 mRNAs were downregulated in OA cartilage (Fold change ≥ 2.0). The qRT-PCR results of six dysregulated lncRNAs were consistent with the microarray data. 106 lncRNAs and 291 mRNAs composed the coding-non-coding gene co-expression network (CNC network). In the 600 top differentially expressed lncRNAs, 48 lncRNAs were predicted to have more than five cis-regulated target genes and up to 530 lncRNAs might regulate their trans target genes through collaboration with transcriptional factor (TF) SP1. The positive correlation between lncRNA uc.343 and predicted target homeobox gene C8 (HOXC8) expression in SW1353 cells treating with interleukin-1 beta confirmed the target prediction to some extent. This study revealed the expression pattern of lncRNAs in OA cartilage and predicted the potential function and targets, which indicated that lncRNAs may be new biomarkers for diagnosis or novel therapeutic targets of OA.

Fu M ，Huang G ，Zhang Z ，Liu J ，Zhang Z ，Huang Z ，Yu B ，Meng F ... -  《-》