Endogenous circadian time genes expressions in the liver of mice under constant darkness

Background The circadian rhythms regulate physiological functions and metabolism. Circadian Time (CT) is a unit to quantify the rhythm of endogenous circadian clock, independent of light influence. To understand the gene expression changes throughout CT, C57BL/6 J mice were maintained under constant darkness (DD) for 6 weeks, and the liver samples were collected starting at 9:00 AM (CT1), and every 4 h in a 24-h cycle (CT5, CT9, CT13, CT17 and CT21). Total RNA was extracted and subjected to RNA-Seq data (deposited as GSE 133342, L-DD). To compare gene oscillation pattern under normal light-dark condition (LD, GSE114400) and short time (2 days) dark-dark condition (S-DD, GSE70497), these data were retried from GEO database, and the trimmed mean of M-values normalization was used to normalize the three RNA-seq data followed by MetaCycle analysis. Results Approximate 12.1% of the genes under L-DD exhibited significant rhythmically expression. The top 5 biological processes enriched in L-DD oscillation genes were mRNA processing, aromatic compound catabolic process, mitochondrion organization, heterocycle catabolic process and cellular nitrogen compound mitotic catabolic process. The endogenous circadian rhythms of clock genes, P450 genes and lipid metabolism genes under L-DD were further compared with LD and S-DD. The oscillation patterns were similar but the period and amplitude of those oscillation genes were slightly altered. RT-qPCR confirmed the selected RNA sequence findings. Conclusions This is the first study to profile oscillation gene expressions under L-DD. Our data indicate that clock genes, P450 genes and lipid metabolism genes expressed rhythmically under L-DD. Light was not the necessary factor for persisting circadian rhythm but influenced the period and amplitude of oscillation genes.

背景 昼夜节律调节生理功能和新陈代谢。昼夜时间（CT）是一种量化内源性生物钟节律的单位，不受光照影响。为了解整个昼夜时间内基因表达的变化，将C57BL/6J小鼠在持续黑暗（DD）环境中饲养6周，并从上午9点（CT1）开始收集肝脏样本，在24小时周期内每隔4小时收集一次（CT5、CT9、CT13、CT17和CT21）。提取总RNA并进行RNA - Seq测序（数据存储为GSE133342，L - DD）。为了比较正常光暗条件（LD，GSE114400）和短时间（2天）暗暗条件（S - DD，GSE70497）下的基因振荡模式，从GEO数据库中重新获取这些数据，并使用M值的加权修剪均值归一化方法对三个RNA - Seq数据进行归一化，然后进行MetaCycle分析。 结果 在L - DD条件下，约12.1%的基因表现出显著的节律性表达。在L - DD振荡基因中富集程度最高的5个生物学过程是mRNA加工、芳香族化合物分解代谢过程、线粒体组织、杂环化合物分解代谢过程以及细胞含氮化合物有丝分裂分解代谢过程。进一步比较了L - DD条件下时钟基因、P450基因和脂质代谢基因的内源性昼夜节律与LD和S - DD条件下的情况。振荡模式相似，但这些振荡基因的周期和振幅略有改变。实时定量聚合酶链反应（RT - qPCR）证实了所选的RNA测序结果。 结论 这是第一项对L - DD条件下振荡基因表达进行分析的研究。我们的数据表明，在L - DD条件下，时钟基因、P450基因和脂质代谢基因呈节律性表达。光照不是维持昼夜节律的必要因素，但会影响振荡基因的周期和振幅。