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Background N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). Methods The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. Results ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. Conclusion ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy.

Disturbances in circadian rhythms are known to affect immune functions. However, the long-term impact of abnormal circadian rhythms on the immune-related functions of the spleen are poorly understood. Hence, we aimed to investigate the immune-related functions of spleen in Per1/Per2 double-knockout (DKO) and wild-type (WT) mice aged 4, 9, and 14 months. Compared to the WT mice, the DKO mice had smaller spleen white pulp (WP) and lymphocyte germinal area, as well as fewer immune cells with age—these differences were especially clear. The spleen lymphocyte mortality, malondialdehyde (MDA) levels, reactive oxygen species (ROS) levels, and ferritin-binding receptor (TFR1) levels were significantly higher in the 14-month-old DKO mice than in WT mice of the same age. Transcriptome analysis showed that most of the differentially expressed mRNAs were enriched in DNA damage repair-related pathways. In DKO mice, spleen cells showed up-regulation of pro-ferroptosis genes, such as Cd36,Atm, and Acsl4, and down-regulation of anti-ferroptosis genes, such as GPX4. We found that long-term abnormalities in the circadian rhythm can induce DNA damage and ferroptosis in mouse spleen.

IntroductionPER1 is a repressor protein involved in regulating circadian rhythm. While obstructive sleep apnea (OSA) is characterized by recurred pauses in breathing caused by the collapse of the upper airways it might be associated with disruption of the circadian clock.ObjectivesThe study aimed to assess PER1 protein in OSA patients and evaluate its association with PSG parameters.MethodsThe study included 40 individuals, who underwent diagnostic polysomnography (PSG) examination. Based apnea-hypopnea index (AHI) patients were divided into groups: control (AHI<5; n=10) and OSA (AHI5; n=30). All participants had their peripheral blood collected in the evening (9:00-10:00 pm) before and in the morning (6:00-7:00 am) after the PSG. PER1 protein concertation measurements were performed using ELISA. Funding: National Science Centre, Poland-2018/31/N/NZ5/03931.ResultsThe control and OSA group were match in sex and age, while differed regarding BMI (p=0.039), desaturation index (p<0.001) and AHI (p<0.001). PER1 protein level was elevated in OSA group compared to control both in the evening (322.384.1vs.208.460.1pg/ml;p<0.001) and morning (314.891.9vs.228.157.3pg/ml;p=0.002). No difference was observed between evening and morning PER1 level (p=0.946). Morning PER1 correlated with AHI (r=0.400; p=0.011), desaturation index (r=0.391;p=0.013), age (r=-0.312;p=0.049) and BMI (r=0.383;p=0.015). In a multiple linear regression model (R2=0.268;p=0.003) morning PER1 protein level was influenced by age (p=0.006) and AHI (p=0.025), while BMI and desaturation index were not significant.ConclusionsOSA patients might suffer from circadian clock disruption, which is mainly associated with the severity of the disorder and age. Further studies are needed as this dysregulation can result in metabolic and mood disorders often observed in this group of patients.

Circadian rhythm disorders caused by genetic or environmental factors lead to decreased male fertility but the mechanisms are poorly understood. The current study reports that the mechanism of Per1/Per2 Double knockout (DKO) reduced the reproductive capacity of elderly male mice. The sperm motility and spermatogenic capacity of male DKO mice were weak. Hormone-targeted metabolomics showed reduced plasma levels of free testosterone in DKO male mice compared with WT male mice. Transcriptomic analysis of testicular tissue showed the down-regulation of testosterone synthesis-related enzymes (Cyp11a1, Cyp17a1, Hsd17b3, Hsd3b1, and Star) in the steroid hormone synthesis pathway. Spermatogenesis genes, Tubd1 and Pafah1b were down-regulated, influencing tubulin dynamics and leading to impaired motility. Seleno-compound metabolic loci, Scly and Sephs2, were up-regulated and Slc7a11 and Selenop were down-regulated. Western-blotting showed that steroid acute regulatory protein (StAR) and p-CREB, PKA and AC1 were reduced in testicular tissue of DKO mice compared to WT. Therefore, Per1/Per2 disruption reduced testosterone synthesis and sperm motility by affecting the PKA-StAR pathway, leading to decreased fertility.

The mechanism underlying premature ovarian insufficiency remains incompletely understood. Here we report that mice with Per1m/m; Per2m/m double mutations display a decrease in female fertility starting approximately at 20 weeks old, with significantly less pups born from 32 weeks old onwards. Histological analysis revealed that a significant reduction of ovarian follicles was observed in the Per1/Per2 mutants compared with the littermate controls examined at 26 and 52 weeks old, while the difference was not statistically significant between the two groups at 3 and 8 weeks old. We further showed that vascular development including the ovarian follicle associated vascular growth appeared normal in the Per1/Per2 mutant mice, although clock genes were reported to regulate angiogenesis in zebrafish. The findings imply that loss-of-function mutations with Per1/Per2 result in a premature depletion of ovarian follicle reserve leading to the decline of reproductive capacity. Summary Sentence Disruption of circadian rhythm or its underlying regulatory network contributes to the premature depletion of ovarian follicle reserve.

Endometrial cancer (EC) is one of the most common gynecologic malignancies, and the incidence rate of night shift among women workers is higher than that in the general population. Circadian rhythm disorder, mainly rhythm gene, is related to various tumor onset, including EC. This study described the sleep/night-shift features of EC patients, explored the mechanism of the circadian clock gene PER and investigated prognostic and functional values of Per1 caused by night shift. A total of 619 subjects were enrolled and divided into two groups according to night-shift duties (rhythm group and control group), analyzed for clinical risk factors and night shift features of endometrial carcinoma. Then samples were randomly selected for sequencing and western blot were performed, and the function of overexpressed PER1 in ishikawa cells was explored. We noticed that severer EC patients experienced night-shift more frequently and with longer durations. A total of 58,174 differentially expressed genes were discovered, mainly rhythm genes and related to up and downstream regulatory genes. Western blot showed that the rhythm group had elevated protein expression of BCAS4, TUBB2B and RSPO4, and decreased expression of PER1 and PER2 in night-shift. In TCGA-EC datasets, PER1 was decreased in the EC patients with a significantly positive correlation with PER2, and higher PER1 expression indicated longer survival, opposite to TUBB2B. The research of overexpressing PER1 gene in EC ishikawa cells found that PER1 can promote apoptosis, expression of TNF-a, IL-6 and PD-1/PD-L1, inhibit the tumor invasion and expression of TUBB2B gene. Together, EC severity was associated with night-shift and rhythm disorders. The rhythm relating factors PER1, TUBB2B and tumor immune factors may regulate the mechanisms of EC onset and progression.

Trazodone is an efficacious atypical antidepressant acting both as an SSRI and a 5HT2A and 5HT2C antagonist. Antagonism to H1-histaminergic and alpha1-adrenergic receptors is responsible for a sleep-promoting action. We studied long-term gene expression modulations induced by chronic trazodone to investigate the molecular underpinning of trazodone efficacy. Rats received acute or chronic treatment with trazodone or citalopram. mRNA expression of growth factor and circadian rhythm genes was evaluated by qPCR in the prefrontal cortex (PFCx), hippocampus, Nucleus Accumbens (NAc), amygdala, and hypothalamus. CREB levels and phosphorylation state were evaluated using Western blotting. BDNF levels were significantly increased in PFCx and hippocampus by trazodone and in the NAc and hypothalamus by citalopram. Likewise, TrkB receptor levels augmented in the PFCx after trazodone and in the amygdala after citalopram. FGF-2 and FGFR2 levels were higher after trazodone in the PFCx. The CREB phosphorylation state was increased by chronic trazodone in the PFCx, hippocampus, and hypothalamus. Bmal1 and Per1 were increased by both antidepressants after acute and chronic treatments, while Per2 levels were specifically augmented by chronic trazodone in the PFCx and NAc, and by citalopram in the PFCx, amygdala, and NAc. These findings show that trazodone affects the expression of neurotrophic factors involved in antidepressant responses and alters circadian rhythm genes implicated in the pathophysiology of depression, thus shedding light on trazodone’s molecular mechanism of action.

Rationale Previous studies have shown that repeated exposure to drugs of abuse is associated with changes in clock genes expression and that mice strains with various mutations in clock genes show alterations in drug-induced behaviors. Objective The objective of this study is to characterize the role of the clock gene mPer1 in the development of morphine-induced behaviors and a possible link to histone deacetylase (HDAC) activity. Methods In Per1 Brdm1 null mutant mice and wild-type (WT) littermates, we examined whether there were any differences in the development of morphine antinociception, tolerance to antinociception, withdrawal, sensitization to locomotion, and conditioned place preference (CPP). Results Per1 Brdm1 mutant mice did not show any difference in morphine antinociception, tolerance development, nor in physical withdrawal signs precipitated by naloxone administration compared to WT. However, morphine-induced locomotor sensitization and CPP were significantly impaired in Per1 Brdm1 mutant mice. Because a very similar dissociation between tolerance and dependence vs. sensitization and CPP was recently observed after the co-administration of morphine and the HDAC inhibitor sodium butyrate (NaBut), we studied a possible link between mPer1 and HDAC activity. As opposed to WT controls, Per1 Brdm1 mutant mice showed significantly enhanced striatal global HDAC activity within the striatum when exposed to a locomotor-sensitizing morphine administration regimen. Furthermore, the administration of the HDAC inhibitor NaBut restored the ability of morphine to promote locomotor sensitization and reward in Per1 Brdm1 mutant mice. Conclusions Our results reveal that although the mPer1 gene does not alter morphine-induced antinociception nor withdrawal, it plays a prominent role in the development of morphine-induced behavioral sensitization and reward via inhibitory modulation of striatal HDAC activity. These data suggest that PER1 inhibits deacetylation to promote drug-induced neuroplastic changes.

Circadian rhythms play an important role in many physiological processes. We have previously reported that no periodic fluctuation in the Bmal1 mRNA is observed in the liver of the chipmunk, a mammalian hibernator, in the hibernation season, suggesting that peripheral circadian clocks are not functional during hibernation. In contrast, the Per2 mRNA levels are transiently increased by elevated body temperature during interbout arousal and showed periodic fluctuations in the hibernation season, suggesting that periodic expression of the Per2 mRNA may be restored during interbout arousal. In the present study, we analyzed Per1 gene expression in the chipmunk liver. The Per1 mRNA showed circadian fluctuations with a peak during the late sleep period in the non-hibernation season and periodic fluctuations with a peak during the early interbout arousal in the hibernation season. In both the non-hibernation and hibernation seasons, Per1 gene expression was phase-advanced relative to Per2 gene expression, and the phase relationship between the two genes was maintained, suggesting that for some genes, periodic gene expression, similar to circadian expression in the non-hibernation season, may be restored during interbout arousal. Interestingly, Per1 gene transcription was differentially activated by BMAL1 in the non-hibernation season and possibly by CREB1 in the hibernation season.

Background Temporomandibular joint osteoarthritis (TMJOA) has a high incidence rate, but its pathogenesis remains unclear. Circadian rhythm is an important oscillation in the human body and influences various biological activities. However, it is still unclear whether circadian rhythm affects the onset and development of TMJOA. Methods We disrupted the normal rhythm of rats and examined the expression of core clock genes in the mandibular condylar cartilage of the jaw and histological changes in condyles. After isolating rat mandibular condylar chondrocytes, we upregulated or downregulated the clock gene Per1 , examined the expression of cartilage matrix-degrading enzymes, tested the activation of the GSK3β/β-CATENIN pathway and verified it using agonists and inhibitors. Finally, after downregulating the expression of Per1 in the mandibular condylar cartilage of rats with jet lag, we examined the expression of cartilage matrix-degrading enzymes and histological changes in condyles. Results Jet lag led to TMJOA-like lesions in the rat mandibular condyles, and the expression of the clock gene Per1 and cartilage matrix-degrading enzymes increased in the condylar cartilage of rats. When Per1 was downregulated or upregulated in mandibular condylar chondrocytes, the GSK3β/β-CATENIN pathway was inhibited or activated, and the expression of cartilage matrix-degrading enzymes decreased or increased, which can be rescued by activator and inhibitor of the GSK3β/β-CATENIN pathway. Moreover, after down-regulation of Per1 in mandibular condylar cartilage in vivo, significant alleviation of cartilage degradation, cartilage loss, subchondral bone loss induced by jet lag, and inhibition of the GSK3β/β-CATENIN signaling pathway were observed. Circadian rhythm disruption can lead to TMJOA. The clock gene Per1 can promote the occurrence of TMJOA by activating the GSK3β/β-CATENIN pathway and promoting the expression of cartilage matrix-degrading enzymes. The clock gene Per1 is a target for the prevention and treatment of TMJOA.