Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-β signaling in chondrocytes.
Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-β signaling in chondrocytes.
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DOI：
10.1016/j.joca.2016.11.007
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发表时间：
2017-06
影响因子：
7
通讯作者：
Lotz MK
中科院分区：
文献类型：
--
作者：
Akagi R;Akatsu Y;Fisch KM;Alvarez-Garcia O;Teramura T;Muramatsu Y;Saito M;Sasho T;Su AI;Lotz MK
通过RNA测序确定昼夜节律（CR）是人类骨关节炎（OA）关节软骨中最失调的通路。本研究检测了培养软骨细胞的昼夜节律性以及CR基因NR 1D 1和BMAL 1在调节软骨细胞功能中的作用。从正常和OA影响的人膝关节软骨中提取RNA（各n=14）。采用定量PCR和免疫组织化学方法检测NR 1D 1和BMAL 1 mRNA和蛋白的表达水平。人软骨细胞同步化，并定期收获，以检查RNA和蛋白质表达的昼夜节律。用NR 1D 1或BMAL 1的小干扰RNA（siRNA）处理软骨细胞，然后进行RNA测序并分析对TGF-β途径的影响。与正常软骨相比，OA中NR 1D 1和BMAL 1 mRNA和蛋白水平显著降低。在培养的人软骨细胞中，观察到NR 1D 1和BMAL 1具有明显的昼夜节律性。在敲低NR 1D 1后观察到BMAL 1表达增加，并且在敲低BMAL 1后观察到NR 1D 1水平降低。用NR 1D 1或BMAL 1 siRNA处理的软骨细胞的RNA测序分别鉴定出330和68个显著不同的基因，这主要影响TGF-β信号通路。OA软骨中的昼夜节律通路失调。干扰培养的软骨细胞中的昼夜节律会影响TGF-β信号传导，这是软骨稳态的中心途径。
Circadian rhythm (CR) was identified by RNA sequencing as the most dysregulated pathway in human osteoarthritis (OA) in articular cartilage. This study examined circadian rhythmicity in cultured chondrocytes and the role of the CR genes NR1D1 and BMAL1 in regulating chondrocyte functions. RNA was extracted from normal and OA-affected human knee cartilage (n=14 each). Expression levels of NR1D1 and BMAL1 mRNA and protein were assessed by quantitative PCR and immunohistochemistry. Human chondrocytes were synchronized and harvested at regular intervals to examine circadian rhythmicity in RNA and protein expression. Chondrocytes were treated with small interfering RNA (siRNA) for NR1D1 or BMAL1, followed by RNA sequencing and analysis of the effects on the TGF-β pathway. NR1D1 and BMAL1 mRNA and protein levels were significantly reduced in OA compared to normal cartilage. In cultured human chondrocytes, a clear circadian rhythmicity was observed for NR1D1 and BMAL1. Increased BMAL1 expression was observed after knocking down NR1D1, and decreased NR1D1 levels were observed after knocking down BMAL1. Sequencing of RNA from chondrocytes treated with NR1D1 or BMAL1 siRNA identified 330 and 68 significantly different genes, respectively, and this predominantly affected the TGF-β signaling pathway. The circadian rhythm pathway is dysregulated in OA cartilage. Interference with circadian rhythmicity in cultured chondrocytes affects TGF-β signaling, which is a central pathway in cartilage homeostasis.