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 ... -  《-》

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 ... -  《-》

Identification of long non-coding RNAs expressed in knee and hip osteoarthritic cartilage.

Long intergenic non-coding RNAs (lincRNAs) are emerging as key regulators in gene expression; however, little is known about the lincRNA expression changes that occur in osteoarthritis (OA). Here we aimed to define a transcriptome of lncRNAs in OA cartilage, specifically comparing the lincRNA transcriptome of knee and hip cartilage. RNA-seq was performed on nucleic acid extracted from hip cartilage from patients undergoing joint replacement surgery because of either OA (n = 10) or because of a neck of femur fracture (NOF; n = 6). After transcript alignment, counts were performed using Salmon and differential expression for ENSEMBL lincRNAs determined using DESeq2. Hip RNA-seq lincRNA expression was compared to a knee dataset (ArrayExpress; E-MTAB-4304). ChIP-seq data from ENCODE was used to determine whether lincRNAs were associated with promoters (plncRNA) or unidirectional enhancer-like regulatory elements (elncRNAs). Our analysis of the hip transcriptome identified 1692 expressed Transcripts Per Million (TPM ≥1) Ensembl lincRNAs, of which 198 were significantly (FDR ≤0.05) differentially expressed in OA vs normal (NOF) cartilage. Similar analysis of knee cartilage transcriptome identified 648 Emsembl lincRNAs with 93 significantly (FDR ≤0.05) differentially expressed in intact vs damaged cartilage. In total, 1834 lincRNAs were expressed in both hip and knee cartilage, with a highly significant correlation in expression between the two cartilages. This is the first study to use RNA-seq to map and compare the lincRNA transcriptomes of hip and knee cartilage. We propose that lincRNAs expressed selectively in cartilage, or showing differential expression in OA, will play a role in cartilage homoeostasis.

Ajekigbe B ，Cheung K ，Xu Y ，Skelton AJ ，Panagiotopoulos A ，Soul J ，Hardingham TE ，Deehan DJ ，Barter MJ ，Young DA ... -  《-》

Long noncoding RNA related to cartilage injury promotes chondrocyte extracellular matrix degradation in osteoarthritis.

Long noncoding RNAs (lncRNAs) play crucial regulatory roles in diverse biologic processes, but knowledge of lncRNAs in osteoarthritis (OA) is limited. The aim of this study was to identify lncRNA expression in articular cartilage and to explore the function of cartilage injury-related lncRNAs (lncRNA-CIR) in OA. To identify lncRNAs specifically expressed in OA cartilage, we compared the expression of lncRNAs in OA cartilage with that in normal cartilage using microarray and quantitative polymerase chain reaction (qPCR) analyses. In OA cartilage, lncRNA-CIR was specifically, differentially, and highly expressed. The function of lncRNA-CIR was determined by silencing and overexpression in vitro. Extracellular matrix (ECM)-related molecules were detected by qPCR, Western blot, and immunofluorescence analyses. Up to 152 lncRNAs were found to be differentially expressed (>8-fold) in OA and normal cartilage (82 lncRNAs more highly expressed and 70 less highly expressed in OA cartilage than in normal cartilage). A specific differentially expressed lncRNA-CIR was selected according to the results of the higher expression in OA cartilage and OA chondrocytes. The expression of lncRNA-CIR increased in chondrocytes with in vitro treatment with interleukin-1β and tumor necrosis factor α. Silencing of lncRNA-CIR by small interfering RNA promoted the formation of collagen and aggrecan and reduced the expression of matrix-degrading enzymes, such as MMP13 and ADAMTS5. The expression of collagen and aggrecan was reduced, whereas the expression of matrix-degrading enzymes was increased, after overexpression of lncRNA-CIR. The results indicate that lncRNA-CIR contributes to ECM degradation and plays a key role in the pathogenesis of OA. We propose that lncRNA-CIR could be used as a potential target in OA therapy.

Liu Q ，Zhang X ，Dai L ，Hu X ，Zhu J ，Li L ，Zhou C ，Ao Y ... -  《-》

Enhanced suppressor of cytokine signaling 3 in arthritic cartilage dysregulates human chondrocyte function.

To determine the expression of suppressor of cytokine signaling 3 (SOCS-3) in human articular chondrocytes and its functional consequences. Chondrocytes were isolated from the cartilage of patients with osteoarthritis (OA), patients with rheumatoid arthritis (RA), and trauma patients and from the healthy cartilage of patients with a femoral neck fracture. The human chondrocyte cell line G6 and primary bovine chondrocytes were used in validation experiments. SOCS-3 messenger RNA (mRNA) expression was measured by quantitative polymerase chain reaction, and SOCS-3 protein levels were determined by Western blotting and immunohistochemical analysis. To ascertain the role of SOCS-3 in the chondrocyte response to interleukin-1β (IL-1β) or lipopolysaccharide (LPS), the expression of SOCS3 was either reduced by small interfering RNA or enhanced by viral transduction. The expression of SOCS-3 mRNA (but not that of SOCS-1 mRNA) was significantly enhanced in chondrocytes obtained from OA cartilage (mean ± SD ΔC(t) 3.4 ± 1.0) and RA cartilage (ΔC(t) 3.4 ± 1.4) compared with cartilage obtained from patients with femoral neck fracture (ΔC(t) 5.3 ± 1.2). The expression of SOCS3 correlated significantly with that of other genes known to be expressed in arthritic chondrocytes, such as MMP13 (r = 0.743), ADAMTS4 (r = 0.779), and ADAMTS5 (r = 0.647), and an inverse relationship was observed with COL2A1 (r = -0.561). Up-regulation of SOCS-3 by IL-1 in G6 chondrocytes and its spontaneous expression in OA chondrocytes were reduced by mithramycin, a specific inhibitor of transcription factor Sp-1. Overexpression of SOCS-3 in bovine chondrocytes reduced IL-1- and LPS-induced nitric oxide production and insulin-like growth factor 1-induced proteoglycan synthesis. Interestingly, a similar impairment of function was observed in OA chondrocytes, which was partially restored by SOCS-3 gene knockdown. This study demonstrated that both SOCS-3 mRNA and SOCS-3 protein are expressed in human arthritic chondrocytes and affect cellular responses involved in cartilage pathology.

van de Loo FA ，Veenbergen S ，van den Brand B ，Bennink MB ，Blaney-Davidson E ，Arntz OJ ，van Beuningen HM ，van der Kraan PM ，van den Berg WB ... -  《-》