Aims

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been reported as the important regulators in osteoarthritis (OA). However, the detailed mechanism is implicated. The aim of this study is to reveal the functional mechanism of lncRNA ARFRP1 and miR-15a-5p in osteoarthritis.

Materials and methods

The expression level of genes was detected by quantitative real time polymerase chain reaction (qRT-PCR) or western blot assay. Cell Counting Kit-8 (CCK-8) was used to assess cell viability. Cell apoptosis rate was analyzed by flow cytometry analysis. Furthermore, Enzyme-linked immunosorbent assay (ELISA) was performed to measure tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β contents. The interaction between miR-15a-5p and ARFRP1 or Toll-like receptor 4 (TLR4) was predicted by miRcode or PITA, and then confirmed by the dual luciferase reporter assay or pull down assay. Besides, NF-κB-driven luciferase activity was determined using NF-κB luciferase reporter assay.

Key findings

ARFRP1 and TLR4 levels were increased and miR-15a-5p level was decreased in OA cartilage tissues and lipopolysaccharides (LPS)-induced chondrocytes. ARFRP1 knockdown inhibited LPS-induced the injury of chondrocytes. Interestingly, miR-15a-5p downregulated by ARFRP1 negatively modulated TLR4 expression through interaction. ARFRP1 mediated LPS-induced the injury of chondrocytes via regulating miR-15a-5p/TLR4 axis. Furthermore, ARFRP1 exerted function by modulation of NF-κB pathway.

Significance

Our findings confirmed that ARFRP1 mediated LPS-induced the injury of chondrocytes through regulating NF-κB pathway by modulation of miR-15a-5p/TLR4 axis, providing theoretical basis for the treatment of OA patients.

中文翻译：

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LncRNA ARFRP1敲低通过调节miR-15a-5p / TLR4轴，通过调节NF-κB途径抑制LPS诱导的软骨细胞损伤。

目的

据报道，长的非编码RNA（lncRNA）和微小RNA（miRNA）是骨关节炎（OA）的重要调节剂。但是，涉及详细的机制。这项研究的目的是揭示lncRNA ARFRP1和miR-15a-5p在骨关节炎中的功能机制。

材料和方法

通过定量实时聚合酶链反应（qRT-PCR）或蛋白质印迹测定法检测基因的表达水平。细胞计数试剂盒8（CCK-8）用于评估细胞活力。通过流式细胞仪分析细胞凋亡率。此外，进行酶联免疫吸附测定（ELISA）以测量肿瘤坏死因子-α（TNF-α），白介素（IL）-6和IL-1β的含量。miRcode或PITA预测了miR-15a-5p与ARFRP1或Toll样受体4（TLR4）之间的相互作用，然后通过双重萤光素酶报告基因测定或下拉测定证实。此外，使用NF-κB荧光素酶报告基因测定法测定了NF-κB驱动的荧光素酶活性。

主要发现

在OA软骨组织和脂多糖（LPS）诱导的软骨细胞中，ARFRP1和TLR4水平升高，而miR-15a-5p水平降低。ARFRP1组合式抑制LPS诱导的软骨细胞损伤。有趣的是，被ARFRP1下调的miR-15a-5p通过相互作用负调节了TLR4的表达。ARFRP1通过调节miR-15a-5p / TLR4轴介导LPS诱导的软骨细胞损伤。此外，ARFRP1通过调节NF-κB途径发挥功能。

意义

我们的研究结果证实，ARFRP1介导的LPS介导的miR-15a-5p / TLR4轴的调节通过调节NF-κB途径，诱导了软骨细胞的损伤，为OA患者的治疗提供了理论依据。