Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst Campus, Neston, Cheshire CH64 7TE, UK.
Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst Campus, Neston, Cheshire CH64 7TE, UK.
Osteoarthritis Cartilage. 2016 Jul;24(7):1263-73. doi: 10.1016/j.joca.2016.01.988. Epub 2016 Feb 4.
Regulation of anabolic and catabolic factors is considered essential in maintaining the homoeostasis of healthy articular cartilage. In this study we investigated the influence of RNA binding proteins (RNABPs) in this process.
Using small interfering RNA (siRNA), RNABP expression was knocked down in SW1353 chondrosarcoma cells and human articular chondrocytes. Gene expression and messenger RNA (mRNA) decay of anabolic (SOX9, Aggrecan) and catabolic (matrix metalloproteinase (MMP)13) factors were analysed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RNA-electromobility shift assays (EMSAs) were used to investigate RNABP interactions with the SOX9 mRNA 3' untranslated region (UTR). Immunohistochemical localisation of MMP13 and the RNABP human antigen R (HuR) was performed in E13.5 and E16.5 mouse embryo sections.
SOX9 mRNA, mRNA half-life and protein expression were increased with siRNA targeting the RNABP tristetraprolin (TTP) in both HACs and SW1353s. TTP knockdown also stimulated aggrecan mRNA expression but did not affect its stability. RNA-EMSAs demonstrated that adenine uracil (AU)-rich elements in the SOX9 mRNA 3'UTR interacted with chondrocyte proteins with three specific elements interacting with TTP. HuR knockdown significantly increased MMP13 expression and also regulated the expression of a number of known transcriptional repressors of MMP13. HuR was ubiquitously expressed within mouse embryos yet displayed regional down-regulation within developing skeletal structures.
This study demonstrates for the first time how RNABPs are able to affect the balance of anabolic and catabolic gene expression in human chondrocytes. The post-transcriptional mechanisms controlled by RNABPs present novel avenues of regulation and potential points of intervention for controlling the expression of SOX9 and MMP13 in chondrocytes.
调控合成代谢和分解代谢因子被认为是维持健康关节软骨内稳态的关键。在本研究中，我们研究了 RNA 结合蛋白（RNABP）在这一过程中的影响。
使用小干扰 RNA（siRNA）敲低 SW1353 软骨肉瘤细胞和人关节软骨细胞中的 RNABP 表达。使用逆转录定量聚合酶链反应（RT-qPCR）分析合成代谢（SOX9、聚集蛋白聚糖）和分解代谢（基质金属蛋白酶（MMP）13）因子的基因表达和信使 RNA（mRNA）衰减。RNA 电泳迁移率变动分析（EMSA）用于研究 RNABP 与 SOX9 mRNA 3'非翻译区（UTR）的相互作用。在 E13.5 和 E16.5 小鼠胚胎切片中进行 MMP13 和 RNA 结合蛋白人类抗原 R（HuR）的免疫组织化学定位。
在 HACs 和 SW1353s 中，针对 RNABP 三丝氨酸脯氨酸（TTP）的 siRNA 可增加 SOX9 mRNA、mRNA 半衰期和蛋白表达。TTP 敲低也刺激聚集蛋白聚糖 mRNA 表达，但不影响其稳定性。RNA-EMSA 表明，SOX9 mRNA 3'UTR 中的腺嘌呤尿嘧啶（AU）丰富元件与软骨细胞蛋白相互作用，其中三个特定元件与 TTP 相互作用。HuR 敲低显著增加 MMP13 表达，还调节了 MMP13 的许多已知转录抑制剂的表达。HuR 在小鼠胚胎中广泛表达，但在发育中的骨骼结构中显示区域下调。
本研究首次证明了 RNABP 如何影响人软骨细胞中合成代谢和分解代谢基因表达的平衡。RNABP 控制的转录后机制为调控软骨细胞中 SOX9 和 MMP13 的表达提供了新的途径和潜在的干预点。
