lncRNA HSP90AA4P通过OPN调控关节相关细胞功能在骨关节炎发病中的分子机制研究

The pathogenesis of osteoarthritis (OA) has not been illustrated clearly. Osteopontin (OPN) is a phosphorylated acidic glycoprotein. We had showed OA patients had high OPN expression in synovium and articular cartilage. Long noncoding RNA (LncRNA) is known as regulatory factor recently. The previous study revealed upregulated transcripts of Wnt signaling pathways and OPN and down-regulated expression of lncRNA HSP90AA4P in OA. SPP1, which codes for osteopontin(OPN), is 87 kb upstream of lncRNA HSP90AA4P (4.15 fold downregulation) and a likely candidate as the cis-regulated target of HSP90AA4P. Both Wnt signaling pathways and transcription factor (TF) SP1 can induce the expression of OPN. LncRNAs can act on their target genes through cis-regulation, which is identified as when the transcription of an lncRNA affects the expressions of its neighbor genes, or long-range trans-regulation in conjunction with other transcription factors. Our previous study demonstrated that Wnt pathway can regulate OPN expression and our preliminary data showed that HSP90AA4P siRNA can upregulate OPN expression and decrease chondrocyte apoptosis. Therefore, we hypothesized that lncRNA HSP90AA4P might play a role in the pathogenesis of OA, it may induce OPN expression by cis-regulation, or long-range trans-regulation in conjunction with TF SP1, or Wnt signaling pathways, further regulate the function of chondrocyte, synoviocyte and osteoclast, and they are appropriate target for therapy. This study aims to: 1) analyze the expression of lncRNA HSP90AA4P and OPN in knee joint tissues, chondrocyte and synovial cell from OA patients and healthy people in this study through a series of methods including qRT-PCR, et al, 2) elucidate the effect and mechanism of lncRNA HSP90AA4P on cell functions via OPN in vitro through a series of methods including RNA-RIP, siRNA, et al, 3) explore the protective and therapeutic effects of lncRNA HSP90AA4P, OPN, and their antibodies or siRNA in vivo through animal experiments. Consequently, this research attempts to reveal pathogenesis of OA, explore new therapeutic target, to provide some new ideas on the treatment of OA in the clinical practice.

骨关节炎(OA)的发病机制尚不十分明了。我们证实骨桥蛋白(OPN)与OA密切相关。文献表明OA软骨中长链非编码RNA(lncRNA) HSP90AA4P低表达、Wnt通路和OPN高表达，并推测HSP90AA4P的候选靶基因是OPN，且可能通过转录因子SP1调控OPN。我们前期发现OA中Wnt通路能调控OPN表达，且预实验显示HSP90AA4P siRNA上调OPN表达并减少软骨细胞凋亡。基于此，我们提出假设：HSP90AA4P可能直接作用于OPN，或通过SP1、再或通过Wnt通路调节OPN表达，进而影响关节相关细胞（软骨细胞、滑膜细胞与破骨细胞）功能，导致OA的发生，且给予靶基因及其siRNA或抗体可能延缓甚至阻止OA的进展。拟从临床标本、细胞实验、裸鼠模型层面，采用RNA-RIP、siRNA、过表达质粒转染等技术，证实上述假设，旨在从新的视觉阐明OA的发病机制，为防治OA提供新靶点。

骨关节炎（OA）是是一种多病因的，最常见的关节退行性疾病。目前发表机制尚不明确，研究表明OA软骨中长链非编码RNA(lncRNA) HSP90AA4P低表达、Wnt通路和OPN高表达，我们推测HSP90AA4P的候选靶基因是OPN我们提出假设：LncRNAHSP90AA4P可能直接作用于OPN，或通过SP1、再或通过Wnt通路调节OPN表达，进而影响关节相关细胞细胞功能。为证实上述推测：通过收集正常和OA患者关节组织，检测LncRNAHSP90AA4P、OPN和相关指标表达，发现LncRNAHSP90AA4P在滑膜和软骨组织中与OPN表达水平和OA病情严重性呈负相关。LncRNAHSP90AA4P在滑膜和软骨细胞中与OPN表达水平和OA病情严重性呈负相关。软骨中MMP-13与软OPN表达呈负相关。运用过表达质粒和siRNA转染OA患者滑膜和软骨细胞，发现LncRNAHSP90AA4P过表达能使软骨和滑膜细胞OPN表达下降，加速软骨和滑膜细胞老化和凋亡，降低软骨和滑膜细胞活性，使软骨细胞肥大，增强滑膜细胞炎症反应。通过软骨细胞研究LncRNAHSP90AA4P与OPN作用实验结果表明LncRNAHSP90AA4P并非通过OPN影响OA滑膜和软骨细胞的功能。动物实验发现膝关节OPN缺乏会加速大鼠膝关节软骨细胞衰老和凋亡，并且加速OA进展。本课题为LncRNAHSP90AA4P作为OA的靶向治疗提供了可能。而且LncRNAHSP90AA4P不通过OPN影响骨关节炎，这为OA发病机制提供了新的研究方向。