Most of the life forms on Earth have gradually evolved an endogenous biological clock under the long-term influence of periodic daily light-dark cycles. This biological clock system plays a crucial role in the orderly progression of life activities. In mammals, central circadian clock is located in the suprachiasmatic nucleus of the hypothalamus and the function of the biological clock relies on a transcription-translation negative feedback loop. As a negative regulator in this loop, the function of CHRONO is less known. To deeply explore the role of the Chrono gene in rhythm entrainment and physiology, we constructed a Chrono gene knockout mouse strain using the CRISPR/Cas9 technology and analyzed its entrainment ability under different T cycles. Running wheel tests and glucose tolerance tests were also performed. The results showed that the period of the endogenous biological clock of Chrono knockout mice was prolonged, and the entrainment rate under the T21 cycle was decreased. In addition, metabolic abnormalities, including weight gain and impaired glucose tolerance, were observed in the non-entrained mice. Overall, this study reveals a crucial role of the Chrono gene in maintaining circadian rhythms and metabolic balance, providing a new perspective for understanding the relationship between the biological clock and metabolism. Further research is needed to fully understand the underlying molecular mechanisms.
Animals ; Mice, Knockout ; Mice ; Circadian Rhythm/genetics* ; Metabolic Diseases/physiopathology* ; Photoperiod ; Male ; Period Circadian Proteins/physiology* ; Light ; Circadian Clocks/physiology*

OBJECTIVE: To investigate the intervention of melatonin (MT) in the expression of circadian genes in patients with pulmonary fibrosis and to analyze the mechanism by which it alleviates the progression of pulmonary fibrosis. METHODS: By utilizing the Gene Expression Omnibus (GEO) database, we identified differentially expressed circadian genes between patients with pulmonary fibrosis and controls. We analyzed the correlation between circadian genes and pulmonary function as well as genes related to pulmonary fibrosis. A bleomycin-induced mouse model of pulmonary fibrosis (BLM group) was constructed to observe the expression differences of PER2 and CRY2 by sequencing and immunohistochemical staining in the BLM group and after MT intervention (BLM+MT group). Hematoxylin and eosin (HE) staining and Masson staining were used to observe the effects of MT on fibrosis. We used Western blot to detect the expression of P-smad2/3 in lung epithelial cells induced by transforming growth factor β (TGF-β). Reverse transcription quantitative real-time PCR technology was employed to investigate the rhythmic expression changes of circadian genes in the control group, TGF-β group, and TGF-β+MT group. Finally, luzindole, a MT receptor antagonist, was used to intervene in TGF-β+MT group, and Western blot was used to explore the receptor dependence of MT in alleviating TGF-β-induced epithelial-mesenchymal transition. RESULTS: (1) Analysis of the GEO dataset (GSE) revealed a negative correlation between circadian genes PER2 and CRY2 and the expression of TGF-β, and a positive correlation with pulmonary function indicators in patients. (2) Transcriptome sequencing analysis of lung tissue in BLM group found that the expression of PER2 and CRY2 was significantly reduced compared with the normal group. Histopathological staining results showed that the lung tissue structure of the normal group was intact and clear, with thin alveolar septa; in the BLM group, there was a large increase in collagen fibers and disordered alveolar structure; compared with the BLM group, the BLM+MT group had reduced collagen fiber proliferation and inflammatory cell infiltration; the expression of PER2 and CRY2 in the BLM group was lower than in the normal group, and the expression in the BLM+MT group was increased compared with the BLM group. (3) In vitro lung epithelial cell experiments with TGF-β intervention showed that compared with the control group, the expression of P-smad2/3 increased in the TGF-β group, and MT intervention inhibited the inducing effect of TGF-β on P-smad2/3, while intervention with the MT receptor antagonist reversed this phenomenon. The results indicated that MT could inhibit the activation of the TGF-β pathway, and this process was dependent on MT receptors. (4) The 48-hour rhythm experiment in lung epithelial cells showed that the mRNA rhythm of PER2 and CRY2 in the TGF-β+MT group was close to 24 hours and showed a trend towards restoring the rhythm of the control group, while the addition of the MT receptor blocker tended to make the rhythm duration and amplitude of both groups approach that of the TGF-β group. CONCLUSION MT, by binding to its receptors, can restore the periodic expression of the circadian genes PER2 and CRY2, thereby inhibiting the activation of the TGF-β classical pathway and suppressing the pathological process of epithelial-mesenchymal transition in pulmonary fibrosis. This finding provides new molecular targets and potential therapeutic strategies for the treatment of pulmonary fibrosis.
Melatonin/pharmacology* ; Animals ; Mice ; Pulmonary Fibrosis/chemically induced* ; Bleomycin ; Humans ; Transforming Growth Factor beta/metabolism* ; Period Circadian Proteins/metabolism* ; Smad3 Protein/genetics* ; Disease Models, Animal ; Lung/pathology* ; Cryptochromes/metabolism* ; Smad2 Protein/genetics* ; Epithelial Cells/metabolism* ; Mice, Inbred C57BL

OBJECTIVETo investigate the effect and regulatory mechanism of clock gene Per1 on the proliferation,apoptosis,migration,and invasion of human oral squamous carcinoma SCC15 cells.

METHODSRNA interference was used to knock down Per1 gene in human oral squamous cell carcinoma SCC15 cell line. Changes of cell proliferation and apoptosis were analyzed by flow cytometry. Transwell assay was carried out to assess cell migration and invasion. Real-time polymerase chain reaction was used to detect the mRNA expressions of Ki-67, murine double minute 2 (MDM2), c-Myc, p53, Bax, Bcl-2, metalloproteinase (MMP)2, MMP9, and vascular endothelial growth factor (VEGF).

RESULTSshRNA-mediated knockdown of Per1 promoted the proliferation, migration and invasion capacity, and inhibited cell apoptosis capacity of SCC15 cells (all P<0.05). Additionally, Per1 knockdown also increased the mRNA expressions of Ki-67, MDM2, Bcl-2, MMP2, and MMP9 and decreased the mRNA expressions of c-Myc, p53, and Bax (all P<0.05); however, the VEGF mRNA expression did not differ significantly after Per1 knockdown (P>0.05).

CONCLUSIONSClock gene Perl can regulate important tumor-related genes downstream such as Ki-67, MDM2, c-Myc, p53, Bax, Bcl-2, MMP2, and MMP9, and the aberrant expression of Per1 can affect tumor cell proliferation,apoptosis,migration and invasion. An in-depth study of Per1 may further clarify the mechanism of tumorigenesis and tumor development and thus provides new effective molecular targets for cancer treatment.

Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Knockdown Techniques ; Humans ; Ki-67 Antigen ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Period Circadian Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA Interference ; Real-Time Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo determine the regulatory effects of the circadian clock gene Per1 on cell cycle-related genes and its influence on the proliferation, apoptosis, cycle, and tumorigenicity in vivo of human oral squamous cell carcinoma SCC15 cells.

METHODSThree groups of the short hairpin RNA (shRNA) of lentivirus recombinant plasmids were designed against the RNA of Per1 and then transfected to the SCC15 cells. The optimum interference group was screened through Western blot and quantitative real-time PCR (qRT-PCR) and assigned as the experimental group. The transfected lentivirus plasmid without an interference effect on any gene was set as the control group (Control-shRNA). Untreated SCC15 cells were set as the blank group. The mRNA expressions of cell cycle-related genes, namely, Per1, p53, Cyclin D1, Cyclin E, Cyclin A2, Cyclin B1, CDK1, CDK2, CDK4, CDK6, p16, p21, Wee1, cdc25, E2F, and Rbl1 in each group were detected through qRT-PCR. The cell proliferation, apoptosis, and cell cycle distribution in each group were evaluated through flow cytometry. The cells of the experimental group and the blank group were subcutaneously inoculated in nude mice to observe tumorigenesis.

RESULTSThree groups of Per1-shRNA lentivirus plasmids were constructed successfully. Among the groups, the Per1-shRNA- I group exhibited the highest interference effect, as indicated by qRT-PCR and Western blot analysis. As such, this group was set as the experimental group. The mRNA expression levels of CyclinD1, CyclinE, CyclinB1, CDK1, and Wee1 gene in the Per1-shRNA-I group were significantly higher than those in the Control-shRNA group and the SCC15 group (P < 0.05). By contrast, the mRNA expression levels of p53, Cyclin A2, p16, p21, and cdc25 in the Per1-shRNA-I group were significantly lower than those in the Control-shRNA group and the SCC15 group (P < 0.05). The mRNA expression levels of each gene between the Control-sLRNA group and the SCC15 group did not significantly differ (P > 0.05). The mRNA expression levels of CDK2, CDK4, CDK6, E2F, and Rb1 did not significantly differed in the three groups (P > 0.05). The proliferation index of the Perl-shRNA-I group was significantly higher than those of the Control-shRNA group and the SCC15 group (P < 0.05). The apoptosis index of the Per1-shRNA-I group was significantly lower than those of the Control-shRNA group and the SCC15 group (P < 0.05). The number of S-phase cells in the Per1-shRNA-I group was significantly lower than those of S-phase cells in the Control-shRNA group and the SCC15 group (P < 0.05). The number of G2/M-phase cells in the Per1-shRNA-I group was significantly higher than those of G2/M-phase cells in the Control-shRNA group and the SCC15 group (P < 0.05). Conversely, the proliferation index, apoptotic index, and cell cycle distribution of the cells in the Control-shRNA group did not significantly differ from those of the SCC15 group (P > 0.05). The tumorigenic ability in vivo was significantly enhanced in the Per1-shRNA-I group (P < 0.05).

CONCLUSIONPer1 is an important tumor suppressor gene. Per1 can regulate a large number of downstream cell cycle-related genes. The alteration of its expression can affect cell cycle progression, proliferation, apoptosis imbalance, and tumorigenic ability in vivo. Further studies on Per1 may elucidate cancer development and provide novel effective molecular targets for cancer treatment.

Animals ; Apoptosis ; Carcinoma, Squamous Cell ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Circadian Clocks ; genetics ; Cyclin D1 ; Humans ; Mice ; Mice, Nude ; Mouth Neoplasms ; Period Circadian Proteins ; genetics ; Plasmids ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Transfection

OBJECTIVETo identify protein-protein interaction partners of PER1 (period circadian protein homolog 1), key component of the molecular oscillation system of the circadian rhythm in tumors using bacterial two-hybrid system technique.

METHODSHuman cervical carcinoma cell Hela library was adopted. Recombinant bait plasmid pBT-PER1 and pTRG cDNA plasmid library were cotransformed into the two-hybrid system reporter strain cultured in a special selective medium. Target clones were screened. After isolating the positive clones, the target clones were sequenced and analyzed.

RESULTSFourteen protein coding genes were identified, 4 of which were found to contain whole coding regions of genes, which included optic atrophy 3 protein (OPA3) associated with mitochondrial dynamics and homo sapiens cutA divalent cation tolerance homolog of E. coli (CUTA) associated with copper metabolism. There were also cellular events related proteins and proteins which are involved in biochemical reaction and signal transduction-related proteins.

CONCLUSIONIdentification of potential interacting proteins with PER1 in tumors may provide us new insights into the functions of the circadian clock protein PER1 during tumorigenesis.

Base Sequence ; Cell Line, Tumor ; Escherichia coli ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Neoplasms ; genetics ; metabolism ; Period Circadian Proteins ; genetics ; metabolism ; Protein Binding ; Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

OBJECTIVEThis study investigates the circadian variation rules of the clock gene Per2 and clock-controlled genes of vascular endothelial growth factor (VEGF), Ki67, c-Myc, and P53 in different stages of carcinogenesis in buccal mucosa carcinoma and their roles in the development of buccal mucosa carcinoma.

METHODSNinety Syrian golden hamsters were housed under. 12 h light/12 h dark cycles. Dimethylbenzanthracene (DMBA) was used to establish the carcinoma model by smearing the golden hamster buccal mucosa. Before DMBA painting and after 6 and 14 weeks, the hamsters were sacrificed at six time points within a period of 24 h (i.e., 4, 8, 12, 16, 20, and 24 h after light onset), and the normal buccal mucosa, precancerous lesions, and cancer tissues were simultaneously obtained. Hematoxylin and eosin stained sections were prepared to observe the canceration of each tissue. Real time polymerase chain reaction was used to detect the mRNA expression of Per2, VEGF, Ki67, c-Myc, and P53. Cosine analysis was employed to determine the circadian-rhythm variations of Per2, VEGF, Ki67, c-Myc, and P53 mRNA expression in terms of median, amplitude, and acrophase.

RESULTSThe expression of Per2, VEGF, P53, and c-Myc mRNA in three different stages appeared with circadian rhythms (P<0.05), whereas the Ki67 mRNA was expressed with circadian rhythm only in normal and precancerous lesion stages (P<0.05). The midline-estimating statistic of rhythms (MESORs) of Per2 and P53 mRNA were significantly down-regulated with the development of cancer (P<0.05), whereas the MESORs of VEGF, c-Myc, and Ki67 mRNA were up-regulated (P<0.05). The amplitude of P53 mRNA significantly decreased with the development of cancer (P<0.05). Moreover, compared with the normal group, the amplitudes of Per2, VEGF, Ki67, and c-Myc mRNA significantly increased in precancerous lesions and cancer tissue (P<0.05). In precancerous stage, the acrophases of Per2, VEGF, and c-Myc mRNA were earlier than that in the normal group, whereas that of Ki67 and P53 mRNA were delayed.

CONCLUSIONThe circadian-rhythm characteristics of the clock gene Per2 and clock-controlled gene expression of VEGF, Ki67, c-Myc, and P53 mRNA have changed with the occurrence and development of carcinoma.

9,10-Dimethyl-1,2-benzanthracene ; Animals ; Carcinogenesis ; Carcinoma, Squamous Cell ; metabolism ; Circadian Rhythm ; Cricetinae ; Mesocricetus ; Mouth Mucosa ; metabolism ; Mouth Neoplasms ; metabolism ; Neoplasm Staging ; Period Circadian Proteins ; genetics ; metabolism ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A

Circadian clocks are the endogenous oscillators that harmonize a variety of physiological processes within the body. Although many urinary functions exhibit clear daily or circadian variation in diurnal humans and nocturnal rodents, the precise mechanisms of these variations are as yet unclear. In the present study, we demonstrate that Per2 promoter activity clearly oscillates in neonate and adult bladders cultured ex vivo from Per2::Luc knock-in mice. In subsequent experiments, we show that multiple local oscillators are operating in all the bladder tissues (detrusor, sphincter and urothelim) and the lumbar spinal cord (L4-5) but not in the pontine micturition center or the ventrolateral periaqueductal gray of the brain. Accordingly, the water intake and urine volume exhibited daily and circadian variations in young adult wild-type mice but not in Per1-/- Per2-/- mice, suggesting a functional clock-dependent nature of the micturition rhythm. Particularly in PDK mice, the water intake and urinary excretion displayed an arrhythmic pattern under constant darkness, and the amount of water consumed and excreted significantly increased compared with those of WT mice. These results suggest that local circadian clocks reside in three types of bladder tissue and the lumbar spinal cord and may have important roles in the circadian control of micturition function.
Animals ; *Circadian Clocks ; Drinking ; Mice ; Organ Specificity ; Periaqueductal Gray/metabolism/physiology ; Period Circadian Proteins/genetics/*metabolism ; Pons/metabolism/physiology ; Spinal Cord/*metabolism/physiology ; Urinary Bladder/innervation/metabolism/*physiology ; Urination

OBJECTIVETo investigate the relationship between genetic variantions of circadian clock genes and risk of breast cancer.

METHODSA case-control study including 406 breast cancer patients and 412 controls was conducted and genes Clock (rs2070062) and Per2 (rs2304672, rs2304669, rs934945) were genotyped by TaqMan real-time PCR. Unconditional logistic regression model was used to analyze the association between the genetic polymorphisms and breast cancer.

RESULTSIndividuals with the rs2304669-TT genotype showed significantly increased breast cancer risk with the OR of 2.33 when compared with the individuals with rs2304669-CC and CT genotypes (P = 0.001). In addition, the three haplotypes containing the risk T allele of rs2304669 were identified to be associated with increased breast cancer risk. However, it was found that rs2304672, rs2070062 and rs934945 polymorphisms were not related with breast cancer risk.

CONCLUSIONSThe locus rs2304669 on Per2 gene is associated with breast cancer risk. Genetic variation of circadian clock genes may increase the susceptibility to breast cancer. Therefore, it may become an important biomarker of susceptibility to breast cancer.

Adult ; Biomarkers, Tumor ; genetics ; Breast Neoplasms ; genetics ; CLOCK Proteins ; genetics ; Carcinoma, Ductal, Breast ; genetics ; Case-Control Studies ; Female ; Genetic Variation ; Humans ; Period Circadian Proteins ; genetics ; Polymorphism, Single Nucleotide ; Risk Factors

OBJECTIVETo detect the expression of Per2 in non-small cell lung cancer (NSCLC), and analyze its clinical significance.

METHODSThe expression of Per2 was determined in 60 NSCLC and 20 normal lung tissues by immunohistochemical assay, and the relationship between Per2 expression and clinicopathological features was analyzed.

RESULTSThe positive expression rates of Per2 in NSCLC and normal lung tissues were 71.7% and 95.0%, respectively (P < 0.05). The expression of Per2 in NSCLC was correlated with pathological differentiation and TNM stage (P < 0.05).

CONCLUSIONThe expression of Per2 in NSCLC is decreased. The negative expression of Per2 may contribute to the development and invasion in NSCLC.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; surgery ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; metabolism ; pathology ; surgery ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Grading ; Neoplasm Staging ; Period Circadian Proteins ; metabolism ; Smoking

OBJECTIVETo investigate the effect of acute heat stress on the day-night circadian gene Per2 mRNA expression in the liver of rats.

METHODSMale Wistar rats were randomly divided into two groups and exposed to heat at 32 degrees celsius; or to a room temperature at 24 degrees celsius; (control). After 7 days of heat exposure, the body temperature was measured by telemetry. The relative weight of the pituitary and adrenal glands was determined after the exposure, and liver Per2 mRNA expression level was detected using RT-PCR.

RESULTSAcute heat stress did not obviously affect body temperature or body weight of the rats. Seven days of heat exposure increased the relative weight of the pituitary and adrenal glands and significantly lowered Per2 mRNA expression level at night.

CONCLUSIONAcute heat stress can interfere with the day-night circadian gene Per2 mRNA expression in rats.

Adrenal Glands ; Animals ; Body Temperature ; Body Weight ; Heat-Shock Response ; genetics ; Liver ; metabolism ; Male ; Organ Size ; Period Circadian Proteins ; genetics ; metabolism ; Pituitary Gland ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar