Huang Yijiang, Chen Daosen, Yan Zijian, Zhan Jingdi, Xue Xinghe, Pan Xiaoyun, Yu Huachen
Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China.
Key Laboratory of Orthopaedics of Zhejiang Province, Wenzhou, China.
Front Physiol. 2021 Mar 11;12:617654. doi: 10.3389/fphys.2021.617654. eCollection 2021.
Osteoarthritis (OA) is a chronic degenerative disease of the joints characterized by articular cartilage damage, subchondral bone remodeling, osteophyte formation, and inflammatory changes. This work aims to investigate the protective role of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) against the apoptosis of chondrocytes.
Chondrocyte cell lines, CHON-001, and ATDC5 were treated with different doses of interleukin-1β (IL-1β) to mimic the inflammatory response during OA pathogenesis. Quantitative real-time polymerase chain reaction was performed to measure MEG3, miR-9-5p, and Krüppel-like factor 4 (KLF4) mRNA expression levels. MEG3 and KLF4 overexpression plasmids, MEG3 shRNA, miR-9-5p mimics, and miR-9-5p inhibitors were transfected into the cells. Cell counting kit-8, wound healing assay, and flow cytometry were conducted to determine cell viability, migration, and apoptotic rate. Dual-luciferase reporter assay was adopted to verify the targeting relationships among MEG3, miR-9-5p, and KLF4. Western blot was used to detect KLF4 protein expression. Enzyme-linked immunosorbent assay was employed to measure the levels of inflammatory factors.
MEG3 expression in chondrocytes was down-regulated by the stimulation of IL-1β, and MEG3 negatively regulated miR-9-5p expression but positively regulated KLF4 expression. MEG3 overexpression strengthened the viability and migration of CHON-001 and ATDC5 cells but restrained the apoptosis and inflammatory response, while MEG3 knockdown had opposite effects. miR-9-5p inhibition or KLF4 overexpression could counteract the effects of MEG3 knockdown on chondrocytes. Besides that, MEG3 was proved to be a molecular sponge for miR-9-5p, and KLF4 was verified as the target of miR-9-5p.
MEG3 can promote chondrocyte proliferation and migration and inhibit apoptosis and inflammation by sponging miR-9-5p to induce KLF4 expression, which provides a promising therapy target for OA treatment.
骨关节炎（OA）是一种慢性关节退行性疾病，其特征为关节软骨损伤、软骨下骨重塑、骨赘形成及炎症改变。本研究旨在探讨长链非编码RNA（lncRNA）母系表达基因3（MEG3）对软骨细胞凋亡的保护作用。
用不同剂量的白细胞介素-1β（IL-1β）处理软骨细胞系CHON-001和ATDC5，以模拟OA发病过程中的炎症反应。采用定量实时聚合酶链反应检测MEG3、miR-9-5p和Krüppel样因子4（KLF4）的mRNA表达水平。将MEG3和KLF4过表达质粒、MEG3短发夹RNA（shRNA）、miR-9-5p模拟物和miR-9-5p抑制剂转染到细胞中。采用细胞计数试剂盒-8、伤口愈合实验和流式细胞术检测细胞活力、迁移能力和凋亡率。采用双荧光素酶报告基因实验验证MEG3、miR-9-5p和KLF4之间的靶向关系。用蛋白质免疫印迹法检测KLF4蛋白表达。采用酶联免疫吸附实验检测炎症因子水平。
IL-1β刺激使软骨细胞中MEG3表达下调，MEG3负向调节miR-9-5p表达，但正向调节KLF4表达。MEG3过表达增强了CHON-001和ATDC5细胞的活力和迁移能力，但抑制了细胞凋亡和炎症反应，而MEG3基因敲低则产生相反的效果。抑制miR-9-5p或过表达KLF4可抵消MEG3基因敲低对软骨细胞的影响。此外，证明MEG3是miR-9-5p的分子海绵，且KLF4被验证为miR-9-5p的靶标。
MEG3可通过充当miR-9-5p的海绵来诱导KLF4表达，从而促进软骨细胞增殖和迁移，抑制细胞凋亡和炎症反应，这为OA治疗提供了一个有前景的治疗靶点。
