LNCPILA, a novel PRMT1 interacting long noncoding RNA, promotes osteoarthritis cartilage degradation via positively regulating the NF-KB pathway

Purpose: Osteoarthritis (OA) is the most prevalent joint disorder characterized by cartilage destruction, synovial inflammation, osteophyte formation and subchondral bone remodeling. Multiple factors including mechanical stress, metabolic alteration and inflammatory mediators are involved in the complex pathogenesis of OA. Interventions targeting these pathogenic factors may contribute to the treatment of OA. Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides (nt) without protein-coding capacities. Emerging evidence has shown that lncRNAs have a wide range of biological functions, and their aberrant expression has been associated with diverse pathological settings, including OA and other joint diseases. The objective of this study was to identify a novel lncRNA targeting inflammatory pathway, and to determine functions and mechanism that are regulated by lncRNA in osteoarthritic chondrocytes. Methods: Articular primary chondrocytes were isolated from human cartilage samples, which were acquired from patients with end-stage knee OA at the time of total knee replacement surgery (n = 27), according to protocols approved by the Ethic Committee of Zhujiang Hospital. Transcriptome sequencing assay was performed to detect lncRNAs expression profiles in primary chondrocytes with inflammatory cytokine (TNF-α 10 ng/mL and IL-1β 10 ng/mL) stimulation for 4 hours. LncRNA expression levels were confirmed by real-time PCR (RT-PCR). Rapid amplification of cloned cDNA ends (RACE) was performed to validate the full length of lncRNA transcript. RNA Fluorescence in situ hybridization (FISH) combined with RT-PCR of the nuclear and cytoplasmic fractionations were used to detect subcellular localization of lncRNA. Interference of lncRNA was conducted using Smart Silencer designed and synthesized in Ribobio (China), while overexpression of lncRNA was performed with pcDNA3.1-lncPILA plasmids. Metabolism of extracellular matrix (ECM) was detected by RT-PCR, western blotting and immunofluorescence in vitro. RNA pull down, RNA immunoprecipitation (RIP) and protein coimmunoprecipitation assay were conducted to explore potential lncRNA-interacting proteins and following detailed mechanism. For in vivo studies, experimental OA was induced in 12-week-old male C57BL/6J mice by destabilization of the medial meniscus (DMM) surgery or by intra-articular (IA) injection (once weekly for 3 weeks) of adenovirus (1 × 109 plaque-forming units in a total volume of 10 μL) expressing lncPILA. Mice were sacrificed 8 weeks after DMM surgery or 8 weeks after the first IA injection, and subjected to histological analyses. Results: Transcriptome sequencing analysis revealed that 1041 annotated or potential lncRNAs were up- or downregulated by over 2-fold in inflammatory cytokine (TNF-α and IL-1β)-stimulated human primary chondrocytes (Student’s t test, false discovery rate [FDR] < 0.2). Among them, 718 lncRNAs showed a decrease in expression, while the remaining lncRNA (323) were highly upregulated in inflamed chondrocytes. Among these lncRNAs, we identified a lncRNA that was highly upregulated by almost 15-fold, which we termed lncPILA. LncPILA that contained 3 exons with 554 nt localized primarily to the nucleus of chondrocytes. Bioinformatics analysis suggested that lncPILA had no protein-coding capacity, which was confirmed by in vitro experiments. Smart Silencer-mediated lncPILA knockdown suppressed the activation of NF-ĸB pathway and attenuated inflammatory cytokine-induced ECM degradation, while lncPILA overexpression promoted the activation of NF-ĸB pathway and deteriorated ECM degradation. Furthermore, ectopic expression of lncPILA in mice joints triggered spontaneous cartilage loss and OA development. Mechanistically, LncPILA interacted with protein arginine methyltransferase PRMT1, promoting PRMT1-mediated arginine methylation of DExH-box helicase 9 (DHX9). Increased arginine methylation of DHX9 enhanced its interactions with NF-ĸB p65, promoting NF-ĸB pathway activation. Conclusions: This is the first report to show that lncPILA is upregulated in inflamed human primary chondrocytes and promotes OA cartilage degradation. LncPILA interacts with PRMT1, which asymmetrically demethylates DHX9 and ultimately promotes the activation of NF-ĸB pathway. Approaches that decrease lncPILA expression or activity have potentials to protect against inflammation-related damage and OA development.