生物钟系统调控的关键基因的鉴定和功能分析将提供对生物钟破坏损害生物体健康的潜在机制的全面理解。初始阶段涉及生物信息学分析,从来自野生型小鼠肝脏组织的三个RNA-seq数据集(GSE184303,GSE114400和GSE199061)中汲取见解,涵盖了一天中的六个不同的时间点。不出所料,鉴定了536个表现出节律表达模式的重叠基因。通过在两个代表性时间点(昼夜节律时间,CT:CT2和CT14)在全局Bmal1基因敲除小鼠(Bmal1-/-)中,肝细胞特异性Bmal1基因敲除小鼠(L-Bmal1-/-),和他们相应的对照组,80个可能由BMAL1调节的基因(称为BMAL1调节基因,BRGs)被识别。这些基因在糖脂代谢中显著富集,免疫反应,和肿瘤发生途径。选择八个BRGs(Nr1d1,Cry1,Gys2,Homer2,Serpina6,Slc2a2,Nmrk1和Upp2)以验证它们在24小时内在对照和L-Bmal1-/-小鼠肝脏中的表达模式。与对照小鼠肝脏中观察到的明显节律模式相反。此外,这些选择的BRGs的表达水平显着降低,不包括Cry1,也在L-Bmal1-/-小鼠肝脏中观察到。染色质免疫沉淀(ChIP)-seq(GSE13505和GSE39860)和JASPAR分析验证了BMAL1与这些基因的启动子和内含子区域的节律结合。此外,条件的发展,从基础脂肪变性到非酒精性脂肪性肝病,和最终的恶性肿瘤,显示人肝脏中Bmal1转录本的持续逐渐下降。将上述BRG与源自人类肝癌数据集的DEGs组合,将Gys2和Upp2鉴定为桥接昼夜节律系统和肝细胞癌(HCC)的潜在节点基因。此外,CCK8和伤口愈合实验表明,人GYS2和UPP2蛋白的过表达抑制了HepG2细胞的增殖和迁移,伴随着肿瘤抑制蛋白p53的表达升高。总之,这项研究系统地鉴定了小鼠肝脏中的节律基因,和可能受BMAL1调控的昼夜节律基因的子集。两个昼夜节律基因,Gys2和Upp2已被提出并验证为推进HCC预防和治疗的潜在候选人。
Identification and functional analysis of key genes regulated by the circadian clock system will provide a comprehensive understanding of the underlying mechanisms through which circadian clock disruption impairs the health of living organisms. The initial phase involved bioinformatics analysis, drawing insights from three RNA-seq datasets (GSE184303, GSE114400, and GSE199061) derived from wild-type mouse liver tissues, which encompassed six distinct time points across a day. As expected, 536 overlapping genes exhibiting rhythmic expression patterns were identified. By intersecting these genes with differentially expressed genes (DEGs) originating from liver RNA-seq data at two representative time points (circadian time, CT: CT2 and CT14) in global Bmal1 knockout mice (Bmal1-/-), hepatocyte-specific Bmal1 knockout mice (L-Bmal1-/-), and their corresponding control groups, 80 genes potentially regulated by BMAL1 (referred to as BMAL1-regulated genes, BRGs) were identified. These genes were significantly enriched in glycolipid metabolism, immune response, and tumorigenesis pathways. Eight BRGs (Nr1d1, Cry1, Gys2, Homer2, Serpina6, Slc2a2, Nmrk1, and Upp2) were selected to validate their expression patterns in both control and L-Bmal1-/- mice livers over 24 h. Real-time quantitative polymerase chain reaction results demonstrated a comprehensive loss of rhythmic expression patterns in the eight selected BRGs in L-Bmal1-/- mice, in contrast to the discernible rhythmic patterns observed in the livers of control mice. Additionally, significant reductions in the expression levels of these selected BRGs, excluding Cry1, were also observed in L-Bmal1-/- mice livers. Chromatin immunoprecipitation (ChIP)-seq (GSE13505 and GSE39860) and JASPAR analyses validated the rhythmic binding of BMAL1 to the promoter and intron regions of these genes. Moreover, the progression of conditions, from basic steatosis to non-alcoholic fatty liver disease, and eventual malignancy, demonstrated a continuous gradual decline in Bmal1 transcripts in the human liver. Combining the aforementioned BRGs with DEGs derived from human liver cancer datasets identified Gys2 and Upp2 as potential node genes bridging the circadian clock system and hepatocellular carcinoma (HCC). In addition, CCK8 and wound healing assays demonstrated that the overexpression of human GYS2 and UPP2 proteins inhibited the proliferation and migration of HepG2 cells, accompanied by elevated expression of p53, a tumor suppressor protein. In summary, this study systematically identified rhythmic genes in the mouse liver, and a subset of circadian genes potentially regulated by BMAL1. Two circadian genes, Gys2 and Upp2, have been proposed and validated as potential candidates for advancing the prevention and treatment of HCC.