The Circadian expression of Piezo1, TRPV4, Connexin26, and VNUT, associated with the expression levels of the clock genes in mouse primary cultured urothelial cells
The Circadian expression of Piezo1, TRPV4, Connexin26, and VNUT, associated with the expression levels of the clock genes in mouse primary cultured urothelial cells
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DOI：
10.1002/nau.23400
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发表时间：
2018-03-01
影响因子：
2
通讯作者：
Koizumi, Schuichi
中科院分区：
文献类型：
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作者：
Ihara, Tatsuya;Mitsui, Takahiko;Koizumi, Schuichi
目的建立小鼠膀胱尿路上皮细胞昼夜节律基因表达的实验模型，研究昼夜节律的功能。方法采用实时定量逆转录聚合酶链式反应(qRT-PCR)和免疫印迹分析方法，对原代培养的野生型(WT)和突变型(CLOCK(19/19))小鼠尿路上皮细胞(UCs)的时钟基因、机械感应器Piezo1和TRPV4以及三磷酸腺苷释放介导分子(ARMM)Cx26和VNUT的基因表达节律进行了研究。用发光计测量了PER2(荧光素酶)敲入小鼠(PER2：：Luc)和PER2：：Luc与Clock(19/19)的UC的生物发光，研究了UC中时钟基因的长期振荡。通过检测时钟短干扰RNA(SiRNA)处理后的基因表达节律，比较时钟点突变和时钟缺陷之间的差异。结果WT小鼠UCs中时钟基因、机械传感器和ARMM的基因表达具有时间依赖性。这些基因产物的丰度也与癌组织中的基因表达节律有关。PER2：：Luc在UC中的生物发光表现出昼夜节律。相比之下，在WT小鼠中观察到的所有基因表达节律在时钟(19/19)小鼠中都被取消。结论时钟基因、机械传感器和ARMM等时间依赖性基因的表达在UC中是可复制的。这些发现表明，UC有可能在时钟基因调控的下尿路昼夜节律功能的研究中取得进展。
AimsTo investigate circadian gene expressions in the mouse bladder urothelium to establish an experimental model and study the functions of the circadian rhythm.MethodsThe gene expression rhythms of the clock genes, mechano-sensors such as Piezo1 and TRPV4, ATP release mediated molecules (ARMM) such as Cx26 and VNUT were investigated in mouse primary cultured urothelial cells (UCs) of wild-type (WT) and Clock mutant (Clock(19/19)) mice using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting analysis. The long-term oscillation of the clock genes in UC was investigated by measuring bioluminescence from UC isolated from Period2(luciferase) knock-in mice (Per2::luc) and Per2::luc with Clock(19/19) using a luminometer. The mRNA expression rhythms after treatment with Clock short interfering RNA (siRNA) were also measured to compare differences between Clock point mutations and Clock deficiency.ResultsThe UCs from WT mice showed the time-dependent gene expressions for clock genes, mechano-sensors, and ARMM. The abundances of the products of these genes also correlated with the mRNA expression rhythms in UCs. The bioluminescence of Per2::Luc in UCs showed a circadian rhythm. By contrast, all the gene expression rhythms observed in WT mice were abrogated in the Clock(19/19) mice. Transfection with Clock siRNA in UCs had the same effect as the Clock mutation.ConclusionsWe demonstrated that the time-dependent gene expressions, including clock genes, mechano-sensors, and ARMM, were reproducible in UCs. These findings demonstrated that UCs have the potential to progress research into the circadian functions of the lower urinary tract regulated by clock genes.