Characterization and function study of circadian rhythm gene CtPRR1 in Carthamus tinctorius L / 药学实践杂志

Objective To explore the effect of circadian rhythm genes on flavonoids biosynthesis in safflower and its molecular mechanism. Methods Based on the transcriptome and metabolomic database of safflower corolla, we screened the circadian rhythm genes that correlate with biosynthesis of flavonoids in safflower. qPCR was used to quantify the expressions of circadian rhythm genes in different flowering stages at different time points in a single day. LC-MS was performed to determine the accumulation of flavonoids. The correlation between them was analyzed as well. Yeast Two-Hybrid experiment was used to verify the interactive proteins of these genes. Results Seven circadian rhythm genes PRR1, PRR2, ELF3, FT, PHYB, GI and ZTL were obtained. PRR1 gene was positively correlated with flavonoids accumulation (r≥0.7). The full length of PRR1 is 3 201 bp, encoding 421 amino acids, which is highly homologous with rice OsPRR73 gene and named as CtPRR1 (GenBank accession number: MW492035). CtPRR1 was mainly expressed in flowers, and the expression level increased in the daytime and declined in the evening gradually. Correspondingly, the content of flavonoids showed an opposite variation. Both of them displayed a circadian rhythm with a negative correlation (r≥−0.7). In addition, 2 heat shock proteins along with 3 AP2 transcription factors interacting with CtPRR1 protein were obtained via Yeast Two-Hybrid experiment. Conclusion CtPRR1 negatively regulated the safflower flavonoids accumulation in a circadian rhythm way, which may be affected by these interacting proteins.

BMAL1 regulates mitochondrial fission and mitophagy through mitochondrial protein BNIP3 and is critical in the development of dilated cardiomyopathy

Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans.

BMAL1 regulates mitochondrial fission and mitophagy through mitochondrial protein BNIP3 and is critical in the development of dilated cardiomyopathy

Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans.

BMAL1 regulates mitochondrial fission and mitophagy through mitochondrial protein BNIP3 and is critical in the development of dilated cardiomyopathy

Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans.

The correlation between PER3 rs2640908 polymorphism and colorectal Cancer in the Japanese population

Background: Colorectal cancer (CRC) is one of the most common cancers in Japan. Many factors influence this cancer, one of which is circadian rhythm disruption. Our research investigated the correlation between singlenucleotide polymorphisms (SNPs) in the Period 3 (PER3) (rs2640908), which is one of the circadian genes, and colorectal cancer in the Japanese population. Methods: The study participants consisted of 121 cases and 197 controls. DNA was extracted from participants' peripheral blood cells, and polymerase chain reaction­restriction fragment length polymorphism analysis (PCRRFLP) was performed to detect genotypes of PER3. Results: Participants with T/T genotype were at lower risk of developing colorectal cancer than participants with C/C genotype (adjusted ORs = 0.32 (95% CI: 0.15­0.63)). When stratified by gender and smoking status, T/T genotype were associated with a decreased susceptibility to cancer in males only (adjusted ORs: 0.23 (95% CI: 0.09­0.59)), T/T genotype were also associated with a decreased susceptibility to cancer among both smokers and non-smokers. Conclusions: A significant association was found between the T allele of PER3 polymorphism and a reduced risk of colorectal cancer, especially in males. Smoking status showed no association with the relationship between PER3 genotype and CRC carcinogenesis (AU)

Role of circadian gene Clock during differentiation of mouse pluripotent stem cells

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.

Effects of circadian gene Bmal1 on proliferation in gastric cancer cells / 实用医学杂志

Objective To investigate the effect of Bmal1 on proliferation in gastric cancer cells and the molecular mechanism, and to provide theoretical and experimental basis for further research targeting circadian therapy for gastric cancer. Methods Applying RNAi technique to silence Bmal1 gene in BGC823 was regarded as experimental group. The normal BGC823 was as control group. The inhibitory effect of the cell line was measured by MTT assay. The mRNA expression of p53, c-Jun and c-Fos was evaluated by real-time RT-PCR. Results Comparing with the data of control group,the inhibitive rates of cell growth in experimental group after 6 h,12 h,24 h were 5.78%(P=0.001),9.20%(P=0.00)and 83.08%(P=0.00)respectively. Down-regulation of Bmal1 decreased p53 (P 0.05). Conclusion RNAi targeting Bmal1 has effects on gastric cancer proliferation by down-regulating p53 mRNA.

Expression of circadian gene Per2 and VEGF in NSCL tissue and its clinical significance / 中国基层医药

Objective To detect the expression of Per2 and VEGF in NSCLC tissue,and analyze its clinical significance.Methods The expression of Per2 and VEGF was measured in 60 NSCLC and 20 normal lung tissue by immunohistochemistry assay,then the relation with clinicopathological parameters was analyzed.Results The positive expression rate of Per2 in NSCLC and in normal lung tissue were 71.7％ and 95.0％ ( x2 =4.683,P ＜0.05 ),while the positive expression rate of VEGF were 58.3％ and 15％ (x2 =11.295,P ＜ 0.01 ).The negative expression of Per2 in NSCLC was correlated with pathological differentiation or TNM stage( x2 =4.413,6.179,all P ＜0.05),while the positive expression of VEGF in NSCLC was correlated with pathological differentiation or lymphatic metastasis (x2 =6.524,P ＜ 0.05 ).The expression of Per2 was negatively correlated with the expression of VEGF in NSCLC (r =-0.381,P ＜ 0.01 ).Conclusion The overexpression of VEGF in NSCLC may contribute to invasion and metastasis,while Per2 may be a beneficial prognosis factor in patients with NSCLC.

Inhibitory Effects of Circadian Gene Per2 on Human Pulmonary Carcinoma Cell / 航天医学与医学工程

Objective To reveal the effects of circadian gene Per2 on growth of human lung cancer cell A549 cell and its mechanism. Methods pcDNA3.1(+)-Per2 was transfected into A549 cells and controlled with the vector pcDNA3.1(+). The expression of Per2 was confirmed with RT-PCR and western blot. The Inhibitory effect of Per2 on cell growth and proliferation was demonstrated with MTT assay and colony-forming assay. Apoptosis was detected with flow cytometry. Results The circadian gene Per2 exhibited a growth-inhibitory and apoptosis-inductory effect on A549 cells. Conclusion The results indicate that Per2 may inhibit A549 cell proliferation,which mechanism might involve cellular apoptosis.