lncRNA NEAT1 的下调与 miR-374b-5p/PGAP1 轴相互作用，加重骨关节炎的发展。
Downregulation of lncRNA NEAT1 interacts with miR-374b-5p/PGAP1 axis to aggravate the development of osteoarthritis.
机构信息
Department of Orthopedics, The First Affiliated Hospital of Soochow University, No.188 Shizi Street, Suzhou, 215000, Jiangsu, China.
Department of Orthopedics, The Third Affiliated Hospital of Shanghai University, The Wenzhou Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou, 325000, China.
出版信息
J Orthop Surg Res. 2023 Sep 11;18(1):670. doi: 10.1186/s13018-023-04147-z.
BACKGROUND
Osteoarthritis (OA), characterized by inflammation and articular cartilage degradation, is a prevalent arthritis among geriatric population. This paper was to scrutinize the novel mechanism of long noncoding RNA (lncRNA) NEAT1 in OA etiology.
METHODS
A total of 10 OA patients and 10 normal individuals was included in this study. Cell model of OA was built in human normal chondrocytes induced by lipopolysaccharide (LPS). An OA Wistar rat model was established through intra-articular injection of L-cysteine and papain mixtures (proportion at 1:2) into the right knee. Quantitative reverse transcription-polymerase chain reaction was employed to ascertain the expression levels of NEAT1, microRNA (miR)-374b-5p and post-GPI attachment to protein 1 (PGAP1), while dual-luciferase reporter experiments were used for the validation of target relationship among them. Cell cycle and apoptosis were calculated by flow cytometry analysis. CCK-8 assay was done to evaluate the proliferative potentials of chondrocytes. The levels of cell cycle-related proteins (Cyclin A1, Cyclin B1 and Cyclin D2) and pro-apoptotic proteins (Caspase3 and Caspase9) were measured by western blotting. Tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 levels were determined via ELISA. Hematoxylin & eosin (HE) Staining was used for pathological examination in OA rats.
RESULTS
Pronounced downregulation of NEAT1 and PGAP1 and high amounts of miR-374b-5p were identified in OA patients, LPS-induced chondrocytes and OA rats. NEAT1 targeted miR-374b-5p to control PGAP1 expression. Loss of NEAT1 or upregulation of miR-374b-5p dramatically accelerated apoptosis, led to the G1/S arrest and promoted the secretion of inflammatory cytokines in LPS-induced chondrocytes, while ectopic expression of PGAP1 exhibited the opposite influences on chondrocytes. Additionally, we further indicated that upregulation of miR-374b-5p attenuated the effects of PGAP1 overexpression on LPS-induced chondrocytes.
CONCLUSIONS
Reduced NEAT1 induces the development of OA via miR-374b-5p/PGAP1 pathway. This suggests that the regulatory axis NEAT1/miR-374b-5p/PGAP1 is a novel and prospective target for OA treatment.
背景
骨关节炎（OA）的特征为炎症和关节软骨降解，是老年人群中常见的关节炎。本文旨在探究长链非编码 RNA（lncRNA）NEAT1 在 OA 发病机制中的新机制。
方法
本研究纳入了 10 名 OA 患者和 10 名正常个体。通过脂多糖（LPS）诱导人正常软骨细胞建立 OA 细胞模型。通过向右侧膝关节内注射 L-半胱氨酸和木瓜蛋白酶混合物（比例为 1:2）建立 OA Wistar 大鼠模型。采用定量逆转录-聚合酶链反应（qRT-PCR）检测 NEAT1、微小 RNA（miR）-374b-5p 和糖基磷脂酰肌醇（GPI）连接蛋白 1（PGAP1）的表达水平，并用双荧光素酶报告实验验证它们之间的靶关系。通过流式细胞术分析计算细胞周期和细胞凋亡。CCK-8 检测软骨细胞的增殖潜能。通过蛋白质印迹法测定细胞周期相关蛋白（Cyclin A1、Cyclin B1 和 Cyclin D2）和促凋亡蛋白（Caspase3 和 Caspase9）的水平。通过酶联免疫吸附试验（ELISA）测定肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）的水平。通过苏木精和伊红（HE）染色对 OA 大鼠进行病理检查。
结果
在 OA 患者、LPS 诱导的软骨细胞和 OA 大鼠中，NEAT1 和 PGAP1 表达明显下调，miR-374b-5p 水平升高。NEAT1 通过靶向 miR-374b-5p 来控制 PGAP1 的表达。缺失 NEAT1 或上调 miR-374b-5p 可显著促进 LPS 诱导的软骨细胞凋亡，导致 G1/S 期阻滞，并促进炎症细胞因子的分泌，而过表达 PGAP1 则对软骨细胞产生相反的影响。此外，我们进一步表明，上调 miR-374b-5p 可减弱 PGAP1 过表达对 LPS 诱导的软骨细胞的影响。
结论
NEAT1 通过 miR-374b-5p/PGAP1 通路诱导 OA 的发生。这表明，NEAT1/miR-374b-5p/PGAP1 调控轴是 OA 治疗的一个新的有前景的靶点。
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