Circadian rhythms help organisms adapt to changes of external environment by regulating energy metabolism and remaining the balance of homeostasis. Numerous researches have proved that the physiological function of liver was precisely controlled by circadian rhythms. Clock, one of core circadian genes, has been demonstrated to regulate the oxidative phosphorylation process of mitochondrial, which provides energy for living cells and acts as one of the hub for apoptosis. However, whether Clock gene regulates mitochondrial apoptosis pathways in liver cells remains less explored. In the present study, we used lentiviral vector to establish a stable AML12 cell lines which were capable of expressing specific shRNA to interfere the expression of Clock gene and investigated the effect of Clock on mitochondrial apoptosis pathways. Herein, we found that the interference of Clock gene could significantly suppress mitochondrial apoptosis pathways by stabilizing mitochondrial membrane potential and inhibiting mitochondria out membrane permeablization, which might be a result of lower expression of BAD and BIM proteins. Moreover, the interference of Clock gene could downregulate the expression of mitochondrial apoptosis factors, i.e. AIF, CYCS, APAF-1 and SMAC, which will suppress the formation of apoptosome and the process of DNA degradation to further inhibit apoptosis process. This work provides an insight on the important role of Clock gene participating in mitochondrial apoptosis pathways of hepatocytes and unveils a probable pathogenesis of how circadian rhythm regulates liver diseases.

中文翻译：

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昼夜节律基因Clock通过调节AML12肝细胞的线粒体膜电位，线粒体膜透化和凋亡因子参与线粒体凋亡途径。

昼夜节律通过调节能量代谢并保持体内平衡，帮助生物适应外部环境的变化。大量研究证明，昼夜节律可精确控制肝脏的生理功能。已经证明，时钟是生物钟的核心基因之一，它可以调节线粒体的氧化磷酸化过程，从而为活细胞提供能量，并充当凋亡的枢纽之一。但是，Clock基因是否调节肝细胞中的线粒体凋亡途径尚待探讨。在本研究中，我们使用慢病毒载体建立了一个稳定的AML12细胞系，该细胞系能够表达特异性shRNA来干扰Clock基因的表达，并研究Clock对线粒体凋亡途径的影响。在这里 我们发现Clock基因的干扰可以通过稳定线粒体膜电位和抑制线粒体膜透化而显着抑制线粒体凋亡途径，这可能是BAD和BIM蛋白表达降低的结果。此外，Clock基因的干扰可能下调线粒体凋亡因子AIF，CYCS，APAF-1和SMAC的表达，从而抑制凋亡小体的形成和DNA的降解，进而抑制细胞凋亡。这项工作提供了对Clock基因参与肝细胞线粒体凋亡途径的重要作用的见解，并揭示了昼夜节律如何调节肝脏疾病的可能发病机理。