PER2 Research Articles

The transcription factor VAX1 in VIP neurons of the suprachiasmatic nucleus impacts circadian rhythm generation, depressive-like behavior, and the reproductive axis in a sex-specific manner in mice.

The suprachiasmatic nucleus (SCN) within the hypothalamus is a key brain structure required to relay light information to the body and synchronize cell and tissue level rhythms and hormone release. Specific subpopulations of SCN neurons, defined by their peptide expression, regulate defined SCN output. Here we focus on the vasoactive intestinal peptide (VIP) expressing neurons of the SCN. SCN VIP neurons are known to regulate circadian rhythms and reproductive function. To specifically study SCN VIP neurons, we generated a novel knock out mouse line by conditionally deleting the SCN enriched transcription factor, Ventral Anterior Homeobox 1 (Vax1), in VIP neurons (Vax1Vip; Vax1fl/fl:VipCre). We found that Vax1Vip females presented with lengthened estrous cycles, reduced circulating estrogen, and increased depressive-like behavior. Further, Vax1Vip males and females presented with a shortened circadian period in locomotor activity and ex vivo SCN circadian period. On a molecular level, the shortening of the SCN period was driven, at least partially, by a direct regulatory role of VAX1 on the circadian clock genes Bmal1 and Per2. Interestingly, Vax1Vip females presented with increased expression of arginine vasopressin (Avp) in the paraventricular nucleus, which resulted in increased circulating corticosterone. SCN VIP and AVP neurons regulate the reproductive gonadotropin-releasing hormone (GnRH) and kisspeptin neurons. To determine how the reproductive neuroendocrine network was impacted in Vax1Vip mice, we assessed GnRH sensitivity to a kisspeptin challenge in vivo. We found that GnRH neurons in Vax1Vip females, but not males, had an increased sensitivity to kisspeptin, leading to increased luteinizing hormone release. Interestingly, Vax1Vip males showed a small, but significant increase in total sperm and a modest delay in pubertal onset. Both male and female Vax1Vip mice were fertile and generated litters comparable in size and frequency to controls. Together, these data identify VAX1 in SCN VIP neurons as a neurological overlap between circadian timekeeping, female reproduction, and depressive-like symptoms in mice, and provide novel insight into the role of SCN VIP neurons.

Risk for Seasonal Affective Disorder (SAD) Linked to Circadian Clock Gene Variants.

Molecular pathways affecting mood are associated with circadian clock gene variants and are influenced, in part, by the circadian clock, but the molecular mechanisms underlying this link are poorly understood. We use machine learning and statistical analyses to determine the circadian gene variants and clinical features most highly associated with symptoms of seasonality and seasonal affective disorder (SAD) in a deeply phenotyped population sample. We report sex-specific clock gene effects on seasonality and SAD symptoms; genotypic combinations of CLOCK3111/ZBTB20 and PER2/PER3B were significant genetic risk factors for males, and CRY2/PER3C and CRY2/PER3-VNTR were significant risk factors for females. Anxiety, eveningness, and increasing age were significant clinical risk factors for seasonality and SAD for females. Protective factors for SAD symptoms (in females only) included single gene variants: CRY1-GG and PER3-VNTR-4,5. Clock gene effects were partially or fully mediated by diurnal preference or chronotype, suggesting multiple indirect effects of clock genes on seasonality symptoms. Interestingly, protective effects of CRY1-GG, PER3-VNTR-4,5, and ZBTB20 genotypes on seasonality and depression were not mediated by chronotype, suggesting some clock variants have direct effects on depressive symptoms related to SAD. Our results support previous links between CRY2, PER2, and ZBTB20 genes and identify novel links for CLOCK and PER3 with symptoms of seasonality and SAD. Our findings reinforce the sex-specific nature of circadian clock influences on seasonality and SAD and underscore the multiple pathways by which clock variants affect downstream mood pathways via direct and indirect mechanisms.

Deciphering the molecular signature of selective neuronal vulnerability in the wake‐promoting lateral hypothalamic area in Alzheimer’s disease: A digital multiplexed gene expression study across Braak stages

AbstractBackgroundSleep/wake disturbance is a common and debilitating symptom of Alzheimer’s disease (AD) that precedes cognitive loss. The lateral hypothalamic area (LHA) is critical in orchestrating the sleep/wake cycle through the neuropeptide orexin and melanin‐concentrating hormone. We previously demonstrated that LHA neurons accumulate AD‐tau from Braak stage (BB) 0 and losses ∼72% of neurons by BB6. Little is known about the molecular changes underlying LHA vulnerability during AD progression. We investigated RNA expression changes in LHA across Braak stages to close this gap.MethodWe dissected LHA from 30um‐thick sections of the brain from healthy control (HC) and subjects at progressive AD stages and extracted RNA using conventional techniques. We ran RNA transcriptomic assay using customized and neuropathology nCounter® (Nanostring) panels. The Wald statistical test was used to compare the groups, and the genes were considered differentially expressed when log2 fold‐change was > = |1| and the p‐value was <0.05.ResultWe found an upregulation of HCRTR1, LDHC, NKX6, and SLC11A1 from early stages (BB2‐3), whereas HCRTR2 RNA levels remained stable. Circadian gene PER2 was downregulated from the intermediate stages (BB4). Gene Ontology analyses detected dysregulation of neuropeptide‐ligand interaction pathways from early AD. Linoleic acid metabolism, fat digestion and absorption, and immune pathways became dysregulated at intermediate stages, while glycolysis and gluconeogenesis pathways were in late stages (BB5‐6). Of note, it is critical to consider that the massive LHA neuronal loss during AD progression may confound the interpretation of changes in late stages because of the few remaining neurons.ConclusionnCounter® is suitable for identifying transcriptome changes in LHA, even at early AD stages. Molecular profiling of LHA across the Braak stage revealed that the earliest changes are associated with an imbalance in neurotransmitter receptors, while the decline in metabolism and increased inflammation marked the intermediate stage. The late‐stage AD is characterized by hypoglycemia and energy deficit. Notably, discrepancies in receptor behavior can inform more effective symptomatic treatment for sleep dysfunction in AD.

Investigation of circadian rhythm gene Per3 in diabetic neuropathy

Purpose Diabetic neuropathy (DN) is a serious complication of diabetes that affects peripheral and autonomic nerves, and it has been linked to irregularities in circadian rhythm. Several studies have demonstrated that disruptions in circadian rhythm and changes in expression of rhythm genes may play a role in the development and progression of diabetes, including the development of DN. Methods In this study, the association between the VNTR polymorphism of the PER3 gene and diabetic neuropathy was investigated. The study included 84 patients with diabetes, 220 patients with diabetic neuropathy, and 218 healthy individuals as the control group. Results Upon analyzing the data from the study, it was found that there was no significant difference in the PER3 VNTR polymorphism between the diabetic neuropathy patients, diabetes and control groups. However, there was a significant difference observed between the control group and the diabetes group, particularly in terms of the 5/5 genotype and 5 alleles. Moreover, a significant difference was observed between the patient group and the control group (p < 0.05). Conclusions In conclusion, first in the world, the relationship between PER3 gene VNTR polymorphism and diabetic neuropathy and diabetes, was investigated. Our results showed that PER3 may be associated with diabetes but not with diabetic neuropathy.

Cloning, tissue distribution, and effects of different circadian rhythms on the mRNA expression levels of circadian clock genes Per1a and Per1b in Phoxinus lagowskii

This study describes the cloning and characterization of Period 1a and Period 1b genes and the analysis of their mRNA and protein expression in Amur minnow (Phoxinus lagowskii) after exposure to different light cycles. The full-length P. lagowskii Per1a and Per1b genes encode proteins consisting of 1393 and 1409 amino acids, and share high homology with the per1 genes of other freshwater fish species. The Per1a and Per1b genes were widely expressed within the brain, eye, and peripheral tissues. The acrophase of the Per1a gene in the pituitary gland occurred during the dark phase at ZT15 (zeitgeber time 15, 12 L: 12 D) and ZT18 (8 L, 16 D), whereas the acrophase of the Per1b gene in the pituitary gland was observed during the light phase. Our study suggests that the expression of Per1a and Per1b in P. lagowskii varied depending on differences in circadian rhythm patterns. The results of our dual-luciferase reporter assays demonstrated that the P. lagowskii Per1b gene enhances the activation of NF-κB. This study is the first to examine the circadian clock gene Per1a and Per1b in the high-latitude fish P. lagowskii, offering valuable insights into the effects of different light periods on this fish species.

Circadian gene Per3 promotes astroblastoma progression through the P53/BCL2/BAX signalling pathway

The key circadian genes, Period1(Per1), Period2(Per2), and Period3(Per3), constitute the mammalian Period gene family. The abnormal expression of Per1 and Per2 is closely related to tumor development, but there are few reports on Per3 and tumorigenesis. This study was conducted to determine whether the abnormal expression of Per3 could influence the progression of astroblastoma. The results indicated that the expression level of Per3 was increased in astroblastoma cells, and the high expression of Per3 was correlated with the poor overall survival time of glioma patients. The role of Per3 in astroblastoma cells was then investigated using two approaches: interference and overexpression. The interference of Per3 inhibited astroblastoma cell proliferation by inducing the cell cycle at the S phase. The interference of Per3 inhibited the migration and invasion of astroblastoma cells, while promoted the astroblastoma cell apoptosis and the expression of the apoptosis genes Cleaved-CASP3, P53, and BAX. The overexpression of Per3 promoted proliferation by affecting the S phase distribution of the astroblastoma cell cycle. The overexpression of Per3 promoted the migration and invasion of astroblastoma cells, while inhibited the astroblastoma cell apoptosis and the expression of apoptosis genes Cleaved-CASP3, P53, and BAX. RNA-seq analysis showed that the interference of Per3 in astrocytoma cells resulted in significant changes in the expression levels of 764 genes. Among the differentially expressed genes enriched in apoptosis-related pathways, the interference of Per3 resulted in significant upregulation of MARCKSL1 expression, in contrast to significant downregulation of SFRP4, EPB41L3, and GPC5 expression. Taken together, our results suggest that Per3 appears to be a pro-cancer gene by altering the proliferation, migration, invasion, and apoptosis of astroblastoma cells. As a result, the Per3 gene may be a promising therapeutic target in the treatment of astroblastoma.

Alterations in Per2, Bcl2 gene expression, and oxidative status in aged rats liver after light pulse at night.

The aging process is characterized by circadian rhythm disruption, in physiology and behavior, which could result from weak entrainment. Light is the most potent cue that entrains the central circadian clock, which in turn synchronizes peripheral clocks in animal tissues. Period 2 (Per2) is one of the clock genes that respond to light. Moreover, oxidative stress could entrain the clock. Therefore, the present work aimed to investigate the role of light when applied late at night on the Per2, B cell lymphoma 2 (Bcl2) gene expression, and oxidative status in aged rats. Aged rats were divided into a control group and a group exposed to a 30-min light pulse applied daily during the subjective night at 5 am (ZT 22) for 4weeks. Per2 and Bcl2 gene expression were quantified in liver tissue. To evaluate oxidative status, Glutathione (GSH), nitric oxide (NO), and malondialdehyde (MDA) were estimated. The light pulse reduced the expression levels of Per2 and Bcl2 mRNA. Although it diminished the levels of malondialdehyde (MDA), nitric oxide (NO) levels were elevated and the glutathione (GSH) levels were declined. In conclusion, the light pulse late at night abolished Per2 mRNA circadian rhythm and reduced its expression in the liver of the aged rat. Similarly, it diminished the anti-apoptotic gene expression, Bcl2. Moreover, it might attenuate oxidative stress through the reduction in MDA levels.

Relationship between circadian rhythm-related genes and extracellular matrix: implications for sleep deprivation

BackgroundSleep deprivation (SD) can lead to the development of various pathological disorders. The extracellular matrix (ECM) compositions and circadian rhythm genes are two pivotal variables of SD. However, their relationships remain undefined during SD.MethodsA mouse SD model was established using a modified multiplatform water environment method. The expression of nerve growth factor (NGF) in mouse hippocampus was detected by an immunofluorescence (IF) method. Protein expression was assessed by western blot, and mRNA analysis was performed by quantitative real-time PCR (qRT-PCR). The differentially expressed genes after SD, the genes associated with stromal score, and gene expression correlation were analyzed by bioinformatic analysis.ResultsThe mouse model of SD was successfully established, as evidenced by the changed morphology, increased Bax and NGF levels, and downregulated Bcl-2 in mouse hippocampus after SD. The differentially expressed genes after SD were closely associated with the ECM compositions. The ECM composition metalloproteinase 9 (MMP9) was under-expressed in mouse hippocampus after SD. The hippocampal MMP9 expression was correlated with the expression levels of circadian genes PER2, PER3, TIMELESS, FBXL3, and NFIL3. PER2 and TIMELESS were upregulated in mouse hippocampus after SD.ConclusionThe current findings suggest a correlation between ECM composition MMP9 and circadian rhythm-related genes PER2 and TIMELESS in mouse hippocampus after SD, providing a novel understanding of the disorders after SD.

Clock gene Per3 deficiency disrupts circadian alterations of gut microbiota in mice.

Clock gene Per3 deficiency disrupts circadian alterations of gut microbiota in mice.

Clock gene Per1 regulates rat temporomandibular osteoarthritis through NF-κB pathway: an in vitro and in vivo study

PurposeTemporomandibular joint osteoarthritis (TMJOA) is a common disease that negatively affects the life quality of human beings. Circadian rhythm acts an important role in life activities. However, whether the clock genes are rhythmic expressed in mandibular condylar chondrocytes, or the clock genes have an effect on the progression of TMJOA remains unknown. In this study, we aim to explore expression of clock genes and regulatory mechanism of TMJOA in rat mandibular condylar chondrocytes.MethodsAfter synchronized by dexamethasone, the expression of core clock genes Per1, Per2, Clock, Cry1, Cry2 and Bmal1 and cartilage matrix degrading factor gene Mmp13 were analyzed in mandibular condylar chondrocytes every 4 h with RT-qPCR. The mandibular condylar chondrocytes were stimulated with IL-1β, and expression of Per1, Mmp13, P65 and p-P65 was assessed by RT-qPCR and Western blot. Sh-Per1 lentivirus was used to assess the effect of clock gene Per1 in IL-1β-induced chondrocytes, and expression of Mmp13, P65 and p-P65 was measured. After establishing a rat TMJOA model using unilateral anterior crossbite (UAC), micro-CT, H & E, Alcian Blue & Nuclear Fast Red and Safranin O & Fast Green, cartilage thickness was utilized to assess the damage of cartilage and subchondral bone. Immunohistochemistry of PER1, MMP13 and P65 was performed in condylar sections.ResultsAll core clock genes and Mmp13 were rhythmically expressed. And Mmp13 expression curve was closed in phase and amplitude with Per1. After stimulation with IL-1β, the expression of MMP13, PER1 and P65 and ratio of p-P65/P65 increased in condylar chondrocytes. After Per1 was down-regulated in condylar chondrocytes, the expression of MMP13 and P65 and ratio of p-P65/P65 decreased. Compared with the condyles of Sham group, the bony parameters of UAC group were significantly worse. The thickness of cartilage in UAC group significantly reduced. The modified Mankin scores and the expression of PER1, MMP13 and P65 in cartilage of UAC group significantly increased compared with Sham group.ConclusionCore clock genes and Mmp13 are rhythmic expressed in rat mandibular condylar chondrocytes. PER1 can regulate the expression of MMP13 through NF-κB pathway in IL-1β-induced mandibular condylar chondrocytes.

STUDY OF THE CIRCADIAN MOLECULAR CLOCK IN PAEDIATRIC PATIENTS WITH SEASONAL ALLERGIC RHINITIS: POTENTIAL APPROACHES TO TREATMENT

Understanding of the relationship between the circadian molecular clock and allergic diseases continues to be studied and may be of practical importance for further selection of optimal treatment. The aim of this study was to evaluate the degree of mRNA expression of the circadian molecular clock genes bmal1 and per1 in buccal epithelial cells and to investigate the effi cacy of protocol treatment in children with seasonal allergic rhinitis, taking into account the time dependence. Material and methods. The study included 20 patients aged 6 to 17 years with a confi rmed diagnosis of seasonal allergic rhinitis (SAR). The control group included 7 healthy children of the same age with no history of allergic or chronic diseases. All patients with SAR were divided into 2 groups of 10 patients each, who received the protocol therapy in the morning (group 1) and in the evening (group 2) for 4 weeks. The expression of circadian molecular clock genes was studied by real-time polymerase chain reaction (real-time PCR) method by sampling the buccal epithelium of the children’s oral cavity in the morning (8:00 am) and in the evening (8:00 pm), with a repeat examination of the samples 1 month after treatment.Statistical analysis was performed using the GraphPad Prism 5.00 computer program (GraphPad Software, Inc., San Diego, CA, USA). The research design was discussed and approved at the meeting of the medical and ethical commission of the Ukrainian Medical and Stomatological Academy, protocol No. 188 dated 11/25/2020, where permission to conduct medical and biological research was issued. The research design was discussed and approved at the meeting of the Medical and Ethical Committee of the Ukrainian Academy of Medicine and Dentistry, Protocol No. 188 dated 25.11.2000, where permission to conduct medical and biological research was granted.The study was conducted in accordance with the research plan of the Pediatrics Department no. 2 of Poltava State MedicalUniversity «Optimization of early diagnosis, treatment and prognosis of the most common childhood diseases», state registration number 0122U001876 (years of implementation 2021-2026) and research work – Research Institute of genetic and immunological bases of development of pathology and pharmacogenetics of PDMU «Development of methods of treatment and prevention of development of pulmonary fi brosis by activation of PPAR-gamma receptors», state registration number 0122U201686 (years of implementation 2023-2025).Results. Morning gene expression in healthy children showed statistically higher levels of per1 (0.780 ± 0.070) compared to bmal1 (0.293 ± 0.074; p&lt;0.001), whereas in the evening the level of the latter (0.697 ± 0.130) was signifi cantly higher than per1 (0.370 ± 0.044; p&lt;0.05). Comparing the levels of per1 in this group, the expression of this gene was higher in the morning (0.780 ± 0.070) than in the evening (0.370 ± 0.044; p&lt;0.001), while the expression of bmal1 was higher at 20:00 (0.697 ± 0.130 vs. 0.293 ± 0.074; p&lt;0.05). In contrast, patients with SAR had a signifi cantly higher level of bmal1 gene mRNA expression (1.036±0.161 vs. 0.293±0.074 in healthy children (p&lt;0.05). Examination of gene expression in children with SAR in the evening revealed that bmal1 values showed similar levels of expression in all participants (0.775 ± 0.163 vs. 0.697 ± 0.130; p&gt;0.05), while the mRNA level of the per1 gene was statistically higher in patients with rhinitis (1.293 ± 0.186; p&lt;0.01).Conclusions. The results obtained indicate a dysregulation of the peripheral molecular circadian clock in the buccal epithelium of patients with SAR. The use of the protocol treatment in children with SAR in the evening demonstrated a better restoration of the expression of the positive and negative regulatory link of the molecular circadian clock.

Mechanism of action of photobiomodulation with light-emitting diode on the glutamine-dependent CT26 cell.

We investigated the mechanism of action of photobiomodulation (PBM) with light-emitting diode (led) 640 nm of glutamine-dependent CT26 cells. Cells were exposed to 0.147-10.979 mW/cm2 of 640 ± 15 nm laser light for 15 min/day for 10 days. Cell proliferation and apoptosis were detected by MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide) and annexin V-FITC assays. mRNA and protein levels of cell proliferation-related genes were measured by RT-PCR and western blotting, respectively. With Gln 7.94 mM, on Day 8 and 10, genes GLUT1, MEK1, ERK2, BCL2, E2F1, HO-1, Ctnnb1, and Per2 was significantly upregulated (p < 0.01) of glutamine addiction. In PBM therapy, compared with the non-illuminated group, 2.17 mW/cm2 can significantly reduce cell apoptosis, the mRNA level of gene mTOR1 was significantly upregulated, and the protein level of raptor of GLUT1 and mTOR1, MEK1/2, and ERK1/2 were upregulated. LED 640 nm inhibits cell apoptosis without increasing cell proliferation by regulating GLUT1, MEK/ERK, and PI3K/AKT/mTOR signals.

The modulatory effect of oolong tea polyphenols on intestinal flora and hypothalamus gene expression in a circadian rhythm disturbance mouse model

The interaction between host circadian rhythm and gut microbes through the gut-brain axis provides new clues for tea polyphenols to improve host health. Our present research showed that oolong tea polyphenols (OTP) improved the structural disorder of the intestinal flora caused by continuous darkness, thereby modulating the production of metabolites related to pyruvate metabolism, glycolysis/gluconeogenesis, and tryptophan metabolism to alleviate the steady-state imbalance. After fecal microbiota transplantation from the OTP group, the single-cell transcriptomic analysis revealed that OTP significantly increased the number of hypothalamus cell clusters, up-regulated the number of astrocytes and fibroblasts, and enhanced the expression of circadian rhythm genes Cry2, Per3, Bhlhe41, Nr1d1, Nr1d2, Dbp and Rorb in hypothalamic cells. Our results confirmed that OTP can actively improve the intestinal environmental state as well as internal/peripheral circadian rhythm disorders and cognitive impairment, with potential prebiotic functional characteristics to notably contribute to host health.

Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates

Background and Objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-de- tecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.&#x0D; Materials and Methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla SHV-1 , bla PER-1 , bla , AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic iden- tification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilu- tions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.&#x0D; Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL pro- ducers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla SHV-1 positive, (40%) bla , (85%) aac (6́)-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.&#x0D; Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detec- tion rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. bauman- nii in these clinical isolates showed frequent commonality of genotypes.

Effect of miR-34a on the expression of clock and clock-controlled genes in DLD1 and Lovo human cancer cells with different backgrounds with respect to p53 functionality and 17β-estradiol-mediated regulation.

The small non-coding RNA miR-34a is a p53-regulated miRNA that acts as a tumour suppressor of colorectal cancer (CRC). Oncogenesis is also negatively influenced by deregulation of the circadian system in many types of tumours with various genetic backgrounds. As the clock gene per2 was recently recognized as one of the target genes of miR-34a, we focused on the miR-34a-mediated influence on the circadian oscillator in CRC cell lines DLD1 and LoVo, which differ in their p53 status. Previously, a sex-dependent association between the expression of per2 and that of miR-34a was demonstrated in CRC patients. Therefore, we also investigated the effect of 17β-estradiol (E2) on miR-34a oncostatic functions. miR-34a mimic caused a pronounced inhibition of per2 expression in both cell lines. Moreover, miR-34a mimic significantly inhibited bmal1 expression in LoVo and rev-erbα expression in DLD1 cells and induced clock gene expression in both cell lines. miR-34a mimic caused a pronounced decrease in sirt1 and cyclin D1 expression, which may be related to the inhibition of proliferation observed after mir-34a administration in DLD1 cells. E2 administration inhibited the migration and proliferation of DLD1 cells. E2 and miR-34a, when administered simultaneously, did not potentiate each other's effects. To conclude, miR-34a strongly influences the expression of components of the circadian oscillator without respect to p53 status and exerts its oncostatic effects via inhibition of sirt1 and cyclin D1 mRNA expression. E2 administration inhibits the growth of DLD1 cells; however, this effect seems to be independent of miR-34a-mediated action. With respect to the possible use of miR-34a in cancer treatment, clock genes can be considered as off-target genes, as changes in their expression induced by miR-34a treatment do not contribute to the oncostatic functions of miR-34a. Possible ambiguous oncogenic characteristics should be taken into consideration in future clinical studies focused on miR-34a.

The Relationship between Clock Genes, Sirtuin 1, and Mitochondrial Activity in Head and Neck Squamous Cell Cancer: Effects of Melatonin Treatment.

The circadian clock is a regulatory system, with a periodicity of approximately 24 h, which generates rhythmic changes in many physiological processes, including mitochondrial activity. Increasing evidence links chronodisruption with aberrant functionality in clock gene expression, resulting in multiple diseases such as cancer. Melatonin, whose production and secretion oscillates according to the light-dark cycle, is the principal regulator of clock gene expression. In addition, the oncostatic effects of melatonin correlate with an increase in mitochondrial activity. However, the direct links between circadian clock gene expression, mitochondrial activity, and the antiproliferative effects of melatonin in cancers, including head and neck squamous cell carcinoma (HNSCC), remain largely unknown. In this study, we analyzed the effects of melatonin on HNSCC cell lines (Cal-27 and SCC9), which were treated with 500 and 1000 µM melatonin. We found that the antiproliferative effect of melatonin is not mediated by the Bmal1 clock gene. Additionally, high doses of melatonin were observed to result in resynchronization of oscillatory circadian rhythm genes (Per2 and Sirt1). Surprisingly, the resynchronizing effect of melatonin on Per2 and Sirt1 did not produce alterations in the oscillation of mitochondrial respiratory activity. These results increase our understanding of the possible antiproliferative mechanisms in melatonin in the treatment of head and neck squamous cell carcinoma and suggest that its antiproliferative effects are independent of clock genes but are directly related to mitochondrial activity.

Low-power red laser and blue LED on circadian gene mRNA levels in human breast cancer cells

Low-power blue light-emitting diode (LED) and red laser have been used for therapeutic proposals based on photobiomodulation (PBM). This effect is trigged after absorption of radiation by photoacceptors, which lead to molecular, cellular and systemic responses. Cryptochromes are involved in circadian cycle control, and associated with development and progression of tumors. Despite such proteins are able to absorb violet-blue lights, there are few data on their participation in PBM. Thus, this work aims to evaluate the effects of radiations emitted by low-power blue LED (470 nm) and red laser (658 nm) on mRNA levels from cryptochromes genes as well as those from involved in their regulation in human breast cancer cells. The MCF-7 and MDA-MB-231 cells were exposed to low-power blue LED (470 nm, 640 J cm−2) and red laser (660 nm, 9 J cm−2), and relative mRNA levels from CRY1, CRY2, PER2, BMAL1 and CLOCK genes were evaluated by reverse transcription-quantitative polymerase chain reaction. The results suggested that exposure to low-power blue LED and red laser do not alter the mRNA levels from cryptochromes genes, and those involved in their regulation, in MCF-7 and MDA-MB-231 human breast cancer cells.

Characteristics of insulin resistance in Korean adults from the perspective of circadian and metabolic sensing genes

BackgroundThe biological clock allows an organism to anticipate periodic environmental changes and adjust its physiology and behavior accordingly.ObjectiveThis retrospective cross-sectional study examined circadian gene polymorphisms and clinical characteristics associated with insulin resistance (IR).MethodsWe analyzed data from 1,404 Korean adults aged 30 to 55 with no history of cancer and cardio-cerebrovascular disease. The population was classified according to sex and homeostasis model assessment of insulin resistance (HOMA-IR) values. Demographics, anthropometric and clinical characteristics, and single nucleotide polymorphisms (SNPs) were analyzed with respect to sex, age, and HOMA-IR values. We used association rule mining to identify sets of SNPs from circadian and metabolic sensing genes that may be associated with IR.ResultsAmong the subjects, 15.0% of 960 women and 24.3% of 444 men had HOMA-IR values above 2. Most of the parameters differed significantly between men and women, as well as between the groups with high and low insulin sensitivity. Body fat mass of the trunk, which was significantly higher in insulin-resistant groups, had a higher correlation with high sensitivity C-reactive protein and hemoglobin levels in women, and alanine aminotransferase and aspartate aminotransferase levels in men. Homozygous minor allele genotype sets of SNPs rs17031578 and rs228669 in the PER3 gene could be more frequently found among women with HOMA-IR values above 2 (p = .014).ConclusionOxidative stress enhanced by adiposity and iron overload, which may also be linked to NRF2 and PER3-related pathways, is related to IR in adulthood. However, due to the small population size in this study, more research is needed.

Cloning, tissue distribution, mRNA expression and functional analysis of circadian clock gene per2 from the high-latitude Amur minnow (Phoxinus lagowskii)

Cloning, tissue distribution, mRNA expression and functional analysis of circadian clock gene per2 from the high-latitude Amur minnow (Phoxinus lagowskii)

Daily Rhythmicity of Muscle-Related and Rhythm Genes Expression in Mackerel Tuna (Euthynnus affinis).

The aim of this study was to investigate the circadian rhythm of muscle-related gene expression in mackerel tuna under different weather conditions. The experiment was carried out under two weather conditions at four sampling times (6:00, 12:00, 18:00, and 24:00) to determine the expression of growth, function, and rhythm genes: white muscle rhythm genes were rhythmic on sunny and cloudy days, except for PER3 and RORA; all functional genes had daily rhythmicity. Red muscle had daily rhythmicity on both sunny and cloudy days; functional genes had daily rhythmicity except for MBNL. The expression levels of the rhythm gene PER1 were determined to be significantly different by independent t-test samples in white muscle at 6:00, 12:00, 18:00, and 24:00 under different weather conditions; the expression levels of the functional genes MBNL and MSTN were both significantly different. In the red muscle, the expression of the rhythm genes PER3, REVERBA, and BMAL1 was determined by independent t-test samples at 6:00, 12:00, 18:00, and 24:00 on cloudy and sunny days; the functional gene MBNL was significantly different. The present study showed that mackerel tuna muscle rhythm genes and functional genes varied significantly in expression levels depending on weather, time of day, and light intensity and that the expression levels of myogenic genes were closely related to clock gene expression. The fish were also able to adapt to changes in light intensity in different weather conditions through positive physiological regulation.