Cry 1 Regulates the Clock Gene Network and Promotes Proliferation and Migration Via the Akt/P53/P21 Pathway in Human Osteosarcoma Cells
54
Citation
32
Reference
10
Related Paper
Citation Trend
Abstract:
The many circadian clock genes buildup a network structure that controls physiological processes such as sleep cycle, metabolism and hormone secretion. A close relationship exists between circadian rhythm and cancers because cell cycle is affected by clock controlled genes (CCGs), including Cyclin D1, Cyclin A, Cyclin E and P21. The abnormal expression of the core circadian clock gene Cryptochrome 1 (Cry1) was found in many types of cancers. However, it is still unclear the exact mechanism of Cry1 dysregulation influences carcinogenesis and progression of cancers. In this study, we investigated the role of Cry1 in regulating proliferation and migration of Hos and U2os human osteosarcoma cells by silencing Cry1 using short hairpin RNA interference. Our data from in vitro and in vivo experiments confirmed that Cry1 knockdown enhanced proliferation and migration of osteosarcoma cells. Then, Cry2, Per1, Per2, Per3, Bmal1 and Clock were found up regulated, while Dec1, Dec2, CK1ε and Npas2 were downregulated at mRNA level. Besides, Akt/P53/P21 signaling was activated after Cry1 silencing and Akt was negatively phosphorylated along with Cry1 expression, while enhanced progression of osteosarcoma cells by Cry1 knockdown was reversed when Akt inhibitor treated. Furthermore, the rescue experiment verified the Akt/P53/P21 was downstream genes of Cry1 to control osteosarcoma progression. Taken together, these findings provide a new insight into how Cry1 regulates clock gene network and promotes proliferation and migration in a Akt dependent manner in human osteosarcoma cells.Keywords:
PER2
Cyclin A
The central circadian clock of the mammalian brain resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the molecular level, the circadian clockwork of the SCN constitutes a self-sustained autoregulatory feedback mechanism reflected by the rhythmic expression of clock genes. However, recent studies have shown the presence of extrahypothalamic oscillators in other areas of the brain including the cerebellum. In the present study, the authors unravel the cerebellar molecular clock by analyzing clock gene expression in the cerebellum of the rat by use of radiochemical in situ hybridization and quantitative real-time polymerase chain reaction. The authors here show that all core clock genes, i.e., Per1, Per2, Per3, Cry1, Cry2, Clock, Arntl, and Nr1d1, as well as the clock-controlled gene Dbp, are expressed in the granular and Purkinje cell layers of the cerebellar cortex. Among these genes, Per1, Per2, Per3, Cry1, Arntl, Nr1d1, and Dbp were found to exhibit circadian rhythms in a sequential temporal manner similar to that of the SCN, but with several hours of delay. The results of lesion studies indicate that the molecular oscillatory profiles of Per1, Per2, and Cry1 in the cerebellum are controlled, though possibly indirectly, by the central clock of the SCN. These data support the presence of a circadian oscillator in the cortex of the rat cerebellum. (Author correspondence: mrath@sund.ku.dk)
PER2
PER1
Cite
Citations (35)
Kidney function follows a strong circadian rhythm that is tightly regulated by clock genes. We previously reported that high salt diet induced dyssynchrony in renal clock gene expression in the cortex and medulla. However, whether changes in other dietary factors pose threats to renal circadian rhythms remain largely unknown. The current study was designed to test the hypothesis that high fat diet disrupts renal clock gene expression with 2 mouse models. First, C57Bl/6J male mice (8 weeks old, n=3) were fed normal fat (NF) or high fat (HF) diet for 20 weeks. During the last 2 weeks of the protocol, mice underwent either time restricted feeding, where food access was allowed only during lights‐off, active period (7 pm–7 am), or sham feeding procedure. Restricted feeding did not cause significant changes in body weight or food consumption. Renal cortex was collected in 4‐hour increments throughout a 24‐hour period and clock gene expression measured by qPCR. We found that Per2 expression has a robust circadian rhythm with a peak expression at zeitgeber time (ZT) 13. We did not observe any significant differences among groups. Second, Period2 Luciferase ( Per2 Luc ) mouse model was utilized to monitor real‐time molecular clock rhythm. Male and female Per2 Luc mice (n=4) were fed NF or HF diet with sham or time restricted feeding. Kidneys were dissected into three parts (cortex, outer and inner medulla) at the end of study and cultured for 3 days to measure bioluminescent rhythms. We found that HF diet lengthened the period of luciferase rhythm by 2 hours (NF: 23.98 hours vs. HF: 26.17 hours, p=0.02) in the cortex. No significant differences were observed in the groups in renal outer medulla or inner medulla. Restricted feeding did not cause any significant changes in any groups. These data suggest that clock gene expression in the kidney follows a circadian rhythm that can be disrupted by chronic high fat diet. Support or Funding Information P01 HL136267 Integrating novel mechanisms controlling sodium excretion and blood pressure UAB School of Medicine AMC21 Circadian Disruption and Susceptibility to Target Organ Damage This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
PER2
Zeitgeber
Medulla
Cite
Citations (0)
The SCN as a site of the circadian clock itself exhibits rhythmicity. A molecular clockwork responsible for the rhythmicity consists of clock genes and their negative and positive transcriptional-translational feedback loops. The authors’ previous work showed that rhythms in clock gene expression in the rat SCN were not yet detectable at embryonic day (E) 19 but were already present at postnatal day (P) 3. The aim of the present study was to elucidate when during the interval E19-P3 the rhythms start to develop in clock gene expression and in clock-controlled, namely in arginine-vasopressin (AVP), gene expression. Daily profiles of Per1, Per2, Cry1, Bmal1, and Clock mRNA and of AVP heteronuclear (hn) RNA as an indicator of AVP gene transcription were assessed in the SCN of fetuses at E20 and of newborn rats at P1 and P2 by the in situ hybridization method. At E20, formation of a rhythm in Per1 expression was indicated, but no rhythms in expression of other clock genes or of the AVP gene were detected. At P1, rhythms in Per1, Bmal1, and AVP and a forming rhythm in Per2 but no rhythm in Cry1 expression were present in the SCN. The Per1 mRNA rhythm was, however, only slightly pronounced. The Bmal1 mRNA rhythm, although pronounced, exhibited still an atypical shape. Only the AVP hnRNA rhythm resembled that of adult rats. At P2, marked rhythms of Per1, Per2, and Bmal1 and a forming rhythm of Cry1, but not of Clock, expression were present. The data suggest that rhythms in clock gene expression for the most part develop postnatally and that other mechanisms besides the core clockwork might be involved in the generation of the rhythmic AVP gene expression in the rat SCN during early ontogenesis.
PER2
PER1
Cite
Citations (71)
PER1
PER2
Cite
Citations (140)
Although studies involving the circadian response to external time cues indicate that the peripheral clocks are dominated mainly by food cues, whether and how changes in the light and food cues affect the circadian rhythm of the renal clock is still largely unknown. In the present study, we found that the circadian phases of Bmal1, Clock, Cry1, Per1, and Per2 were altered differently by the stimuli of food and light cues in the kidney. After the individual reversal of the light-dark (LD) cycle for 7 days, Per1 displayed a 4-h phase delay, whereas the peak phases of Bmal1, Clock, Cry1 and Per2 almost remained the same as those in the control condition. With regard to the feeding-induced circadian resetting of the renal clock, we found that the resetting processes of clock genes could not be completed within 7 days, suggesting a weak synchronization effect of the food cue on the renal circadian clock. Moreover, the reentrainment of the clock genes was greatly enhanced after the reversal of both the feeding schedule and the LD cycle. Noticeably, the phases of Per1 and Clock were shifted most rapidly by 12 h within 3 days after the simultaneous reversal of the feeding schedule and the LD cycle, whereas their peak phases were only shifted by 4 h and 8 h, respectively, on the 7th day after the individual reversal of the feeding schedule. Thus Per1 and Clock may play important roles in the light-induced resetting of the circadian rhythms in the kidney.
PER1
PER2
Oscillating gene
Cite
Citations (35)
A comprehensive understanding of the equine circadian clock involves the evaluation of circadian clock gene expression. A non-invasive and effective method for detecting equine clock gene expression has yet to be established. Currently, research surrounding this area has relied on collecting tissue biopsies or blood samples that can often be costly, time consuming and uncomfortable for the animal.Five mares were individually stabled under a light-dark (LD) cycle that mimicked the external environmental photoperiod during a time of year corresponding with the vernal equinox. Hair follicles were collected every 4 h over a 24-h period by plucking hairs from the mane. RNA was extracted and quantitative (q) PCR assays were performed to determine temporal expression patterns for the core clock genes; ARNTL, CRY1, PER1, PER2, NR1D2 and the clock controlled gene, DBP.Repeated measures ANOVA for the clock gene transcripts PER1 and PER2 and the clock controlled gene, DBP, revealed significant variation in expression over time (p < .05, respectively). Cosinor analysis confirmed a significant 24-h temporal component for PER1 (p = .002) and DBP (p = .0033) and also detected rhythmicity for NR1D2 (p = .0331).We show that the extraction of RNA from equine hair follicle cells can identify the circadian 24 h oscillations of specific clock genes and a clock-controlled gene and therefore provide a valuable non-invasive method for evaluating the equine peripheral circadian clock. This method will serve as a useful tool for future evaluations of equine circadian rhythms and their response to environmental changes.
PER2
PER1
Oscillating gene
Cite
Citations (18)
The synchronization of the master clock to photic cues is associated with a rapid induction of Per1, which plays an important role in initiating light-induced circadian resetting. However, the transcriptional mechanisms of clock gene expression in food-entrainable peripheral clocks have not been fully assessed. To understand how food cues might entrain a mammalian peripheral clock, we examined the responses in the expression of clock genes in rat livers to different feeding stimuli. The food-entrainable liver clock is more flexible than the light-entrainable SCN clock and can be reset rapidly at any time of day. A 30 min feeding stimulus was sufficient to significantly induce the expression of Per2 and Dec1 within 1 h and alter the transcript levels and circadian phases of other selected clock genes (Bmal1, Cry1, Per1, Per3, Dec2, and Rev-erba) in the liver clock at longer time intervals. Moreover, among the examined clock genes, Per2 was most sensitive to food cues, which could be significantly induced by a minimal amount of food. Furthermore, in contrast to the other hepatic clock genes, the feeding reversal-induced 12 h phase shift of Per2 could be rapidly and consistently accomplished, regardless of the shift of the light/dark cycle. In conclusion, the feeding-induced resetting of the circadian clock in the liver is associated with the acute induction of Per2 and Dec1 transcription, which may serve as the main and secondary input regulators that initiate this feeding-induced circadian resetting. (Author correspondence: azwfu2003@yahoo.com.cn)
PER2
Oscillating gene
PER1
HSF1
Cite
Citations (44)
Aims To investigate circadian gene expressions in the mouse bladder urothelium to establish an experimental model and study the functions of the circadian rhythm. Methods The 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 Δ19 /Δ 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 Δ19 /Δ 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. Results The 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 s rhythms observed in WT mice were abrogated in the Clock Δ19 /Δ 19 mice. Transfection with Clock siRNA in UCs had the same effect as the Clock mutation. Conclusions We 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.
PER2
Cite
Citations (21)
PER2
Rostral ventrolateral medulla
Cite
Citations (53)
Mammalian retina harbours a self-sustained circadian clock able to synchronize to the light : dark (LD) cycle and to drive cyclic outputs such as night-time melatonin synthesis. Clock genes are expressed in distinct parts of the tissue, and it is presently assumed that the retina contains several circadian oscillators. However, molecular organization of cell type-specific clockworks has been poorly investigated. Here, we questioned the presence of a circadian clock in rat photoreceptors by studying 24-h kinetics of clock and clock output gene expression in whole photoreceptor layers isolated by vibratome sectioning. To address the importance of light stimulation towards photoreceptor clock properties, animals were exposed to 12 : 12 h LD cycle or 36 h constant darkness. Clock, Bmal1, Per1, Per2, Cry1, Cry2, RevErbα and Rorβ clock genes were all found to be expressed in photoreceptors and to display rhythmic transcription in LD cycle. Clock genes in whole retinas, used as a reference, also showed rhythmic expression with marked similarity to the profiles in pure photoreceptors. In contrast, clock gene oscillations were no longer detectable in photoreceptor layers after 36 h darkness, with the exception of Cry2 and Rorβ. Importantly, transcripts from two well-characterized clock output genes, Aanat (arylalkylamine N-acetyltransferase) and c-fos, retained sustained rhythmicity. We conclude that rat photoreceptors contain the core machinery of a circadian oscillator likely to be operative and to drive rhythmic outputs under exposure to a 24-h LD cycle. Constant darkness dramatically alters the photoreceptor clockwork and circadian functions might then rely on inputs from extra-photoreceptor oscillators.
PER2
PER1
Oscillating gene
Darkness
Melanopsin
Cite
Citations (45)