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Our previous study indicated that Clock gene could affect the thrombotic potential. In this present study, we examined the differential expression of proteins in Clock knockdown mice's plasma, including α1-antitrypsin as a potential target. The proteins were examined in AML12 cells with Clock gene being knocked down to confirm the differential expressions. RNAs of those cells were extracted every 3 h in 24 h. The transcriptional levels of α1-antitrypsin (Serpina1a) and fibronectin (Fn1) were analyzed with the least-squares fit of a 24-h cosine function by single cosinor method, but no circadian rhythm was determined in neither of these genes. The expressions of α1-antitrypsin and fibronectin in Clock knockdown cells were found upregulated in both transcriptional and translational levels. Then, we applied ELISA assay to detect the concentration of activated protein C in Clock knockdown plasma and found a risen concentration. Fibronectin was reported to play a role in inhibiting the platelet aggregation, while activated protein C was demonstrated to inhibit PAI-1. All these results indicated that downregulation of the Clock gene in the circulatory system might have effects on PAI-1 by upregulating α1-antitrypsin, and might play some roles in platelet aggregation by increased fibronectin. [ABSTRACT FROM AUTHOR]

Colorectal carcinoma (CRC) is one of the most prevalent types of malignancy-associated mortality of the world. Recently, the studies about over-expression circadian locomotor output cycles kaput gene will promote CRC progression and inhibit tumor cell apoptosis in vitro through the AKT-pathway (an antiapoptotic pathway have been reported). However, it remains to be studied the molecular mechanism of proliferation in CRC. In our present study, we attempt to study the clock gene which inhibits proliferation of CRC CT26 cells through p53-dependent pathway when the clock gene is suppressed in the CRC cell line CT26. Lentiviral vector binds to RNA target sequence, knocking down clock genes in CT26 cells with short hairpin RNA. For detecting their proliferation rates, we used CCK8 assay, plate clone formation assay, Western blot and mouse tumorigenicity assay. The results revealed that knocking down the clock gene has a negative effect on CT26 cell proliferation, depicted that low expression of clock gene reduced cylinD1 and c-myc expression, improved P21 and P53 expression in vitro and inhibited the growth of tumor in vivo. In conclusion, while silencing the clock gene can depress CT26 cell growth through p53-dependent pathway and c-myc. [ABSTRACT FROM AUTHOR]

Social isolation usually affects the physical and mental health of the human and produces depression, whether it may relate with circadian rhythm still remains to be investigated. Two groups of mice were employed to this experiment to continuously monitoring their activities in a dark environment. Immunofluorescence was used to show the expression of the circadian clock gene in two groups of mice. The results demonstrate that the social isolation leads to decrease of mouse activities, phase advance of rhythm relatively and down-regulated of clock gene expression. We conclude that social isolation can affect the physiological function and the normal physiological rhythm of mice, which may be related to depression. [ABSTRACT FROM AUTHOR]

Transactivator (Tat), a regulatory protein of HIV-1, plays a very important role in HIV-1 infection by promoting the rapid replication of HIV. Research surrounding Tat protein function has mainly focused on inhibition of the immune system, promotion of growth of vascular endothelial cells, and nervous system damage. To date, very little research has addressed the role of Tat in circadian rhythms. Previous studies in our lab have found that the concentration of Tat protein in HIV patients’ blood was positively correlated with patients’ sleep quality and melatonin concentrations. In this study, we applied a Tat expression plasmid in mice. Result demonstrated that the locomotor activities of mice and the concentration of melatonin were significantly increased. Alternatively, the expression ofClockgene was markedly decreased. On the other hand, the expression of theCry1gene was significantly increased, while the expression ofBmal1andPer1genes exhibited no significant difference. [ABSTRACT FROM PUBLISHER]

Gravity heavily influences living organisms on earth including their circadian rhythm, which is fundamentally important for coordinately physiology in organisms as diverse as cyanobacteria, fungus and humans. Numerous researches have revealed that microgravity in outer space can affect circadian rhythm of astronauts and rodent animals, but the mechanism remains unknown. Using rotary cell culture system to simulate microgravity environment, we investigated the role of simulated microgravity in regulating the circadian rhythm of NIH3T3 cells. Our experiments found that simulated microgravity can not only influence the mRNA level of some core circadian genes, but also modify the circadian rhythm ofPer1andPer2synchronized after phorbol myristate acetate treatment. Remarkably, MEK/ERK pathway was transiently activated after a 2-h simulated microgravity treatment, with a significant upregulation ofKras,Raf1and p-ERK1/ERK2. Moreover, U0126, a selective inhibitor of MEK/ERK pathway, could disrupt the circadian rhythm ofPer1andPer2synchronized after simulated microgravity treatment. Together, our results unveil that simulated microgravity could act like a zeitgeber to influence the circadian rhythm of NIH3T3 by acting on MEK/ERK pathway, indicating that MEK/ERK pathway may act as a bridge which connects cells mechanotransduction pathway and circadian rhythm regulation. [ABSTRACT FROM PUBLISHER]

Circadian locomotor output cycles kaput (Clock) gene is a core gene in the circadian rhythm system that is involved in cancer cell proliferation. However, the molecular mechanism ofClockgene participate in the cancer cell proliferation is unclear. Previous studies demonstrated that cell proliferation could be regulated by the canonical Wnt pathway (also known as the Wnt/β-catenin pathway), and the Wnt/β-catenin pathway had a relation with the circadian system. To investigate whether theClockgene affects the proliferation of breast cancer cell by regulating the expression ofβ-catenin, we knocked down theClockexpression of mouse breast cancer cells (4T1) by RNA interference. Then detected their proliferation rates using CCK8 assay and the expression of theβ-cateningene by real-time PCR and Western blot. The results showed that the proliferation of theClockknocked down 4T1 cells is slower than the control. The expression level ofβ-cateninof these 4T1 cells is reduced. Our study showed thatClockgene knocked down inhibiting the proliferation of the 4T1 cells, probably by suppressing the expression of β-catenin. [ABSTRACT FROM PUBLISHER]

J. Neurochem. (2010) 115, 353-362. Abstract Protracted opiate withdrawal can extend for months of disrupted hormonal circadian rhythms. We examined rodent behaviors and these circadian disturbances in hormone and peptide levels as well as brain clock gene expression during 60 days of protracted withdrawal. Our behavioral tests included open field, elevated plus maze, and sucrose preference tests at 36 h, 10, 30, and 60 days after stopping chronic morphine. At these four assessment points, we collected samples every 4 h for 24 h to examine circadian rhythms in blood hormone and peptide levels and brain expression of rPER1, rPER2, and rPER3 clock genes. Decreased locomotor activity and elevated adrenocorticotropic hormone and melatonin levels persisted for 2 months after morphine withdrawal, but corticosterone was elevated only at 36 h and 10 days after withdrawal. Orexin levels were high at 36 h after withdrawal, but then reversed during protracted withdrawal to abnormally low levels. Beta-endorphin (β-EP) levels showed no differences from normal. However, circadian rhythms were blunted for all of these hormones. Corticosterone, adrenocorticotropic hormone, and orexin blunting persisted at least for 60 days. The blunted circadian rhythm of β-EP and melatonin recovered by day 60, but the peak phase of β-EP was delayed about 8 h. Blunted circadian rhythms and reduced expression of rPER1, rPER2, and rPER3 persisted at least for 60 days in the suprachiasmatic nucleus, prefrontal cortex, nucleus accumbens core, central nucleus of the amygdala, Hippocampus, and ventral tegmental area. Circadian rhythms of rPER1 in the nucleus accumbens shell and basolateral nucleus of the amygdala and of rPER2 in the central nucleus of the amygdala were reversed. Disrupted circadian rhythms of rPER1, rPER 2, and rPER3 expression in reward-related brain circuits and blunted circadian rhythms in peripheral hormones and peptides may play a role in protracted opiate withdrawal and contribute to relapse. [ABSTRACT FROM AUTHOR]

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. • Identification of 536 rhythmic genes in mouse liver from three datasets. • Identification of 80 circadian genes through omics integration analysis. • Identification of Gys2 and Upp2 as node genes between circadian rhythms and HCC. • GYS2 and UPP2 overexpression attenuates cell proliferation and migration in HCC. [ABSTRACT FROM AUTHOR]