Wang Yueshu, Yu Tao, Jin Hui, Zhao Changfu, Wang Yang
Department of Hand Surgery, China-Japan Union Hospital of Jilin University, Changchun, China.
Department of Orthopedics, China-Japan Union Hospital of Jilin University, Changchun, China.
Cell Physiol Biochem. 2018;45(2):733-743. doi: 10.1159/000487165. Epub 2018 Jan 31.
BACKGROUND/AIMS: Osteoarthritis (OA) is one of the most common chronic degenerative diseases. Many studies have demonstrated the role of microRNAs (miRNAs) in OA; however, the role of miR-302b in OA remains elusive. The aim of this study was to identify the role of miR-302b in LPS-induced injury in chondrocytes.
Human OA chondrocytes (C28/12 cell line) were transfected with miR-302b inhibitor and miR-302b mimic to investigate the effects of miR-302b expression on chondrocyte apoptosis and inflammation, and to identify the miR-302b target proteins.
LPS treatment of chondrocytes significantly reduced cell viability and increased apoptotic rate. LPS treatment also increased the expression of inflammatory cytokines compared to control. miR-302b was up-regulated in LPS-induced chondrocytes. miR-302b was either suppressed or overexpressed in LPS-induced chondrocytes by transient transfection. miR-302b mimic transfection accelerated the effects of LPS on cell viability, apoptosis and inflammation. Of contrast, miR-302b inhibition represented a reverse effect. Dual luciferase activity demonstrated that Smad3 is a direct target for miR-302b and its expression was negatively regulated by miR-302b. In addition, miR-302b inhibition suppressed inflammation in LPS treated chondrocytes by up-regulating Smad3 expression. Moreover, LPS induced down-regulation of Notch and mTOR signaling pathway-related protein expressions, and miR-302b inhibition increased the expressions of Notch and mTOR signaling pathway-related proteins. We further found that miR-302b negatively regulated Notch2 levels through direct targeting its 3'UTR.
These results suggest that miR-302b suppression may function as a protector in suppressing the inflammation during the development and progression of OA by up-regulating the target Smad3 expression.
背景/目的：骨关节炎（OA）是最常见的慢性退行性疾病之一。许多研究已证实微小RNA（miRNA）在OA中的作用；然而，miR-302b在OA中的作用仍不清楚。本研究旨在确定miR-302b在脂多糖（LPS）诱导的软骨细胞损伤中的作用。
将miR-302b抑制剂和miR-302b模拟物转染到人OA软骨细胞（C28/12细胞系）中，以研究miR-302b表达对软骨细胞凋亡和炎症的影响，并鉴定miR-302b的靶蛋白。
LPS处理软骨细胞显著降低细胞活力并增加凋亡率。与对照组相比，LPS处理还增加了炎性细胞因子的表达。在LPS诱导的软骨细胞中miR-302b上调。通过瞬时转染在LPS诱导的软骨细胞中抑制或过表达miR-302b。miR-302b模拟物转染加速了LPS对细胞活力、凋亡和炎症的影响。相比之下，miR-302b抑制则产生相反的效果。双荧光素酶活性表明Smad3是miR-302b的直接靶标，其表达受到miR-302b的负调控。此外，miR-302b抑制通过上调Smad3表达抑制LPS处理的软骨细胞中的炎症。此外，LPS诱导Notch和mTOR信号通路相关蛋白表达下调，而miR-302b抑制增加了Notch和mTOR信号通路相关蛋白的表达。我们进一步发现miR-302b通过直接靶向其3'非翻译区（UTR）负调控Notch2水平。
这些结果表明，miR-302b抑制可能通过上调靶标Smad3表达，在OA发生和发展过程中发挥抗炎保护作用。
