Department of Rheumatology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China.
Department of Respiratory and Critical Care Medicine, Shanghai Sixth People's Hospital, Shanghai Jiaotong University, Shanghai, China.
Int J Rheum Dis. 2021 Mar;24(3):402-410. doi: 10.1111/1756-185X.14053. Epub 2021 Jan 27.
To clarify the interaction of microRNA-320c (miR-320c) and mitogen-activated protein kinase 1 (MAPK1), and to investigate the effects of miR-320c on articular chondroctye proliferation and apoptosis.
Lentiviral expression vectors were constructed and dual luciferase assays containing MAPK1 3'-untranslated regions (3'-UTRs) were performed. Small hairpin RNA (shRNA) was utilized to modulate MAPK1 expression. The messenger RNA and protein expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting respectively. Cell Counting Kit-8 and flow cytometry were conducted to detect the proliferation and apoptosis of Human Chondrocyte-articular (HC-a) cells. Besides that, the influences of miR-320c and MAPK1 on MAPK pathway activation were also evaluated.
Our data identified MAPK1 as a direct target gene of miR-320c, and miR-320c can negatively regulate MAPK1 expression by directly binding to MAPK1 3'-UTR in HC-a cells. Further functional study displayed that miR-320c overexpression and MAPK1 shRNA significantly suppressed the proliferation of HC-a cells and promoted cell apoptosis. Meanwhile, MAPK1 shRNA could attenuate miR-320c inhibitor promotive effects on HC-a cell proliferation and reverse its inhibitory effect on cell apoptosis. MAPK1 overexpression could rescue the inhibitory effect of miR-320c on HC-a cell proliferation, and weaken the accelerating effect of miR-320c on cell apoptosis. However, neither miR-320c or MAPK1 shRNA regulate the expression of c-JUN, JNK and c-Fos.
miR-320c inhibits articular chondrocyte proliferation and induces apoptosis by targeting MAPK1, suggesting that miR-320c perhaps participates in the pathogenesis of osteoarthritis and acts as a potential target for the therapeutic treatment of osteoarthritis.
阐明 microRNA-320c（miR-320c）与丝裂原活化蛋白激酶 1（MAPK1）的相互作用，并研究 miR-320c 对关节软骨细胞增殖和凋亡的影响。
构建慢病毒表达载体，并进行包含 MAPK1 3'-非翻译区（3'-UTR）的双荧光素酶报告基因实验。利用小发夹 RNA（shRNA）来调节 MAPK1 的表达。通过定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹法分别检测信使 RNA 和蛋白质的表达水平。细胞计数试剂盒-8 和流式细胞术检测人软骨细胞-关节（HC-a）细胞的增殖和凋亡。此外，还评估了 miR-320c 和 MAPK1 对 MAPK 通路激活的影响。
我们的数据确定了 MAPK1 是 miR-320c 的直接靶基因，miR-320c 可以通过直接结合 HC-a 细胞中的 MAPK1 3'-UTR 来负调控 MAPK1 的表达。进一步的功能研究显示，miR-320c 过表达和 MAPK1 shRNA 显著抑制 HC-a 细胞的增殖并促进细胞凋亡。同时，MAPK1 shRNA 可以减弱 miR-320c 抑制剂对 HC-a 细胞增殖的促进作用，并逆转其对细胞凋亡的抑制作用。MAPK1 过表达可以挽救 miR-320c 对 HC-a 细胞增殖的抑制作用，并减弱 miR-320c 对细胞凋亡的加速作用。然而，miR-320c 或 MAPK1 shRNA 均不调节 c-JUN、JNK 和 c-Fos 的表达。
miR-320c 通过靶向 MAPK1 抑制关节软骨细胞增殖并诱导凋亡，提示 miR-320c 可能参与骨关节炎的发病机制，并可作为骨关节炎治疗的潜在靶点。
