Sun Hao, Huang Zhiyu, Wu Peihui, Chang Zongkun, Liao Weiming, Zhang Zhiqi
Cell Physiol Biochem. 2018;51(2):909-923. doi: 10.1159/000495392. Epub 2018 Nov 22.
BACKGROUND/AIMS: Cyclin-dependent kinase 6 (CDK6) regulates inflammatory response and cell differentiation. This study sought to determine whether CDK6 and miR-320c co-regulate chondrogenesis and inflammation.
Utilizing quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC), CDK6 and miR-320c expression were assessed in a micromass culture of human bone mesenchymal stem cells that underwent chondrogenesis in vitro as well as in chondrocytes from E16.5 mouse forelimbs. Normal chondrocytes were transfected with miR-320c mimic, miR-320c inhibitor, or CDK6-siRNA. Luciferase reporter assay results confirmed that miR-320c directly targets CDK6 by interacting with the 3'-untranslated region (3'-UTR) of its mRNA. qRT-PCR, Western blotting, and Cell Counting Kit-8 were subsequently used to evaluate the effects of miR-320c overexpression and CDK6 inhibition on inflammatory factor expression, as well as to investigate the effects of NF-kB and MAPK signaling pathway activation on IL-1β-induced chondrocyte inflammation.
Our results show that miR-320c expression increased during the middle stage and decreased during the late stage of hBMSC chondrogenic differentiation. In contrast, CDK6 expression decreased during the middle stage and increased during the late stage of hBMSC chondrogenic differentiation. Moreover, CDK6 expression increased in severe OA cartilage and in hypertrophic chondrocytes of mouse forelimbs at E16.5. Results of the luciferase reporter assay showed that miR-320c modulated CDK6 expression by binding to the 3'-UTR of its mRNA. miR-320c overexpression and CDK6 inhibition repressed IL-1β-induced expression of inflammatory factors and regulated the NF-kB signaling pathway.
CDK6 and miR-320c co-regulate hBMSC chondrogenesis and IL-1β-induced chondrocyte inflammation through the NF-kB signaling pathway, suggesting that miR-320c and CDK6 inhibitors can be used to repress catabolism in human chondrocytes.
背景/目的：细胞周期蛋白依赖性激酶6（CDK6）调节炎症反应和细胞分化。本研究旨在确定CDK6和miR-320c是否共同调节软骨生成和炎症。
利用定量实时PCR（qRT-PCR）和免疫组织化学（IHC），评估人骨髓间充质干细胞微团培养物在体外软骨生成过程中以及E16.5小鼠前肢软骨细胞中CDK6和miR-320c的表达。用miR-320c模拟物、miR-320c抑制剂或CDK6-siRNA转染正常软骨细胞。荧光素酶报告基因检测结果证实，miR-320c通过与其mRNA的3'-非翻译区（3'-UTR）相互作用直接靶向CDK6。随后使用qRT-PCR、蛋白质印迹法和细胞计数试剂盒-8评估miR-320c过表达和CDK6抑制对炎症因子表达的影响，并研究NF-κB和MAPK信号通路激活对IL-1β诱导的软骨细胞炎症的影响。
我们的结果表明，在人骨髓间充质干细胞软骨分化的中期，miR-320c表达增加，而在后期降低。相反，在人骨髓间充质干细胞软骨分化的中期，CDK6表达降低，而在后期增加。此外，在严重骨关节炎软骨和E16.5小鼠前肢肥大软骨细胞中，CDK6表达增加。荧光素酶报告基因检测结果表明，miR-320c通过结合其mRNA的3'-UTR调节CDK6表达。miR-320c过表达和CDK6抑制可抑制IL-1β诱导的炎症因子表达并调节NF-κB信号通路。
CDK6和miR-320c通过NF-κB信号通路共同调节人骨髓间充质干细胞软骨生成和IL-1β诱导的软骨细胞炎症，提示miR-320c和CDK6抑制剂可用于抑制人软骨细胞的分解代谢。
