Huiling Guo

Association of the 54‐nucleotide repeat polymorphism of hPer3 with heroin dependence in Han Chinese population

Circadian clock genes have the function of producing circadian rhythm. They are also implicated in the origin or development of many diseases such as cancers and neuropsychiatric diseases. The purpose of this study is to determine whether the 54‐nucleotide repeat polymorphism of hPer3, one of the circadian clock genes, associates with heroin dependence. DNA samples were obtained from 209 Chinese heroin‐dependent subjects and 249 Chinese healthy controls. The 54‐nucleotide repeat polymorphism was detected by polymerase chain reaction and DNA agarose gel electrophoresis. The frequency of four‐repeat allele was significantly higher (χ2= 10.64, P = 0.001; corrected for multiple tests, P = 0.003) in the mixed gender heroin‐dependent subject group (four repeat: 0.89, five repeat: 0.11) than in the mixed gender control group (four repeat: 0.81, five repeat: 0.19); the frequency of four‐repeat allele was also significantly higher (χ2= 10.00, P = 0.002; corrected for multiple tests, P = 0.006) in the male heroin‐dependent subject group (four repeat: 0.89, five repeat: 0.11) than in the male control group (four repeat: 0.81, five repeat: 0.19); for females, no significant trend was observed with the 54‐nucleotide repeat polymorphism between the heroin‐dependent subject group and the control group. Our results suggest that the 54‐nucleotide repeat polymorphism of hPer3 significantly associates with heroin dependence at the allele frequency level and may be a potential risk factor for the development of heroin dependence.

Circadian renal rhythms influenced by implanted encapsulated hANP-producing cells in Goldblatt hypertensive rats

Renal excretion in experimental hypertensive rats implanted with encapsulated human atrial natriuretic peptide (hANP)-producing cells is circadian periodic. Chinese hamster ovary (CHO) cells transfected with the plasmid hANP-cDNA were encapsulated in biocompatible polycaprolactone capsules for intraperitoneal implantation into two-kidney, one-clip (2K1C) hypertensive rats. During a 12:12 light–dark cycle, as compared to control CHO cells, the implantation of encapsulated hANP-producing CHO cells was associated with an increase in the net excretion of water, sodium and potassium, and with a reversal of the advanced circadian phases related to renovascular hypertension in 2K1C rats. The increase in blood pressure postimplantation was delayed, and increases in renal blood flow, glomerular filtration rate, sodium output, urinary excretion and urinary cyclic GMP concentrations were also found. Implantation of encapsulated hANP-producing cells affects circadian rhythms in kidney excretion functions of 2K1C rats, and may be useful for the treatment of cardiovascular disease.

Circadian blood pressure and heart rate characteristics in haemorrhagic Vs ischaemic stroke in Chinese people

To compare the circadian variation of blood pressure (BP) between patients with intra-cerebral haemorrhage (ICH) and with cerebral infarction (CI), around-the-clock BP measurements were obtained from 89 hypertensive patients with ICH, from 63 patients with CI and from 16 normotensive volunteers. The single and population-mean cosinor yielded individual and group estimates of the MESOR (Midline Estimating Statistic Of Rhythm, a rhythm-adjusted mean value), circadian double amplitude and acrophase (measures of extent and timing of predictable daily change). Comparison shows that without any difference in BP MESOR, the circadian amplitude of systolic (S) BP was larger in ICH than CI patients (P<0.001), and both groups differed from the healthy volunteers in BP MESOR and pulse pressure (P<0.001) and in the circadian amplitude of SBP (P<0.005). The smaller population circadian amplitude of diastolic (D) BP of the ICH group (P=0.042) is likely related to a larger scatter of individual circadian acrophases in this group as compared with that in the other two groups, an inference supported by a smaller day–night ratio of DBP for ICH vs CI patients (P=0.007). Heart rate (HR) variability, gauged by the standard deviation (SD), was decreased in both patient groups as compared with that in healthy controls, more so among ICH than CI patients (P=0.025). Thus, patients with ICH had a higher incidence of abnormal circadian characteristics of BP than patients with CI, the major differences relating to a larger circadian amplitude of SBP, a smaller HR-SD, and a larger incidence of odd circadian acrophases of DBP.

Downregulation of Clock in circulatory system leads to an enhancement of fibrinolysis in mice

As a main component of circadian genes, clock plays not only an important role in circadian rhythm but also in the regulation of many physiological systems. The dysfunction of clock genes is associated with the development of various disorders. Many studies have investigated the association between clock genes and blood coagulation and the fibrinolytic system. The present study was designed to investigate the effect of downregulation of circulatory Clock on blood coagulation and fibrinolysis at the initial stage of active phase in male mice. Downregulation of the expression of the Clock gene by siRNA and, subsequently, its effect on the thrombotic potential and the expression of relative coagulative and/or fibrinolytic factors were investigated. It was found that the Clock interfered mice were less liable to thrombosis and showed prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) at Zeitgeber time (ZT) 15. Meanwhile, these mice also showed an increase in factor VII (FVII) and a decrease in thrombomodulin (TM) and plasminogen activator inhibitor 1 (PAI-1) at ZT 15 at both transcriptional and translational levels. PT, APTT and mRNA expressions of fvii, tm and pai-1 were analyzed with the least-squares fit of a 24-h cosine function by single cosinor method; no circadian rhythm was determined in PT and APTT, and a higher amplitude of fvii in the Clock RNAi group was found with a circadian phase shift, while lower amplitudes of tm and pai-1 were found in the Clock RNAi group with nearly no phase shift. All these results suggest that downregulation of the Clock gene in circulatory system has an effect on factors involved in both blood coagulation and fibrinolysis resulting in an enhancement in mice. This may be considered as an indication that Clock regulates thrombotic homeostasis through the fibrinolytic system.

Nicotinamide adenine dinucleotide: a possible circadian zeitgeber functioning in nontranscription oscillation

Emerging evidences have shown that circadian rhythm can also be regulated by a nontranscriptional mechanism. In this study, we investigated the in vitro rhythmic characters of peroxiredoxin2 (PRX2) and nicotinamide adenine dinucleotide (NAD) in red blood cells (RBCs) and/or in heparin anticoagulated plasma under different light conditions. Our results showed that NAD had a free-running rhythm in accordance with that of PRX2 in RBCs, and both rhythms were liable to light entrainment. A decreased expression of PRX2 in RBCs and a notable phase delay of locomotor activity were found in mice intravenously injected with NAD, suggesting that NAD in nontranscriptional oscillating systems could receive the light zeitgeber signal and influence the expression of PRX2. NAD may also act as a nonphotic zeitgeber, which could make an advanced phase shift of per2, entraining the transcriptional oscillating system to make these two oscillating systems synchronized in a very short time. This zeitgeber might be of great significance as a mediator between these two oscillating systems.

The influence of L-arginine on circadian rhythm and circadian period genes

To study the effect of L-arginine on circadian system, ICR mice and AR4-2J cells were treated with L-arginine. The locomotor activity of the mice intraperitoneally injected with a high dose of L-arginine was observed. We also detected effects of arginine on Per1 and Per2 gene expressions in AR4-2J cells. Double-plotted actograms were applied to analyze the locomotor activity of the mice, and reverse transcription-polymerase chain reaction (RT-PCR) was used in analysis of the expression of the major circadian genes Per1 and Per2 in AR4-2J cells. The results presented here indicate that the intraperitoneal injection of L-arginine would strongly disrupt the circadian rhythm of locomotor activity in mice. While the co-culture of L-arginine would also disturb the circadian rhythm of Per1 and Per2 genes which were induced by PMA in AR4-2J cells, the expression levels of both genes greatly decreased.

Circadian locomotor output cycles kaput affects the proliferation and migration of breast cancer cells by regulating the expression of E-cadherin via IQ motif containing GTPase activating protein 1

The circadian rhythm regulates numerous physiological activities, including sleep and wakefulness, behavior, immunity and metabolism. Previous studies have demonstrated that circadian rhythm disorder is associated with the occurrence of tumors. Responsible for regulating a number of functions, the Circadian locomotor output cycles kaput (Clock) gene is one of the core regulatory genes of circadian rhythm. The Clock gene has also been implicated in the occurrence and development of tumors in previously studies. The present study evaluated the role of the Clock gene in the proliferation and migration of mouse breast cancer 4T1 cells, and investigated its possible regulatory pathways and mechanisms. It was reported that downregulation of Clock facilitated the proliferation and migration of breast cancer cells. Further investigation revealed the involvement of IQ motif containing GTPase activating protein 1 (IQGAP1) protein expression in the Clock regulatory pathway, further influencing the expression of E-cadherin, a known proprietor of tumor cell migration and invasion. To the best of our knowledge, the present study is the first to report that Clock, acting through the regulation of the scaffolding protein IQGAP1, regulates the downstream expression of E-cadherin, thereby affecting tumor cell structure and motility. These results confirmed the role of Clock in breast cancer tumor etiology and provide insight regarding the molecular avenues of its regulatory nature, which may translate beyond breast cancer into other known functions of the gene.

Knock-down the clock gene can lead to colon carcinoma CT26 cell proliferation arrest through p53-dependent pathway and c-myc gene

ABSTRACT Colorectal carcinoma (CRC) is one of the most prevalent types of malignancy-associated mortality of the world. Recently, the studies about over-expression circadian locomotor output cycles kaput gene will promote CRC progression and inhibit tumor cell apoptosis in vitro through the AKT-pathway (an antiapoptotic pathway have been reported). However, it remains to be studied the molecular mechanism of proliferation in CRC. In our present study, we attempt to study the clock gene which inhibits proliferation of CRC CT26 cells through p53-dependent pathway when the clock gene is suppressed in the CRC cell line CT26. Lentiviral vector binds to RNA target sequence, knocking down clock genes in CT26 cells with short hairpin RNA. For detecting their proliferation rates, we used CCK8 assay, plate clone formation assay, Western blot and mouse tumorigenicity assay. The results revealed that knocking down the clock gene has a negative effect on CT26 cell proliferation, depicted that low expression of clock gene reduced cylinD1 and c-myc expression, improved P21 and P53 expression in vitro and inhibited the growth of tumor in vivo. In conclusion, while silencing the clock gene can depress CT26 cell growth through p53-dependent pathway and c-myc.

Clock gene affect the noncanonical NF-κB pathway via circadian variation of Otud7b

Abstract The circadian system regulates many important aspects of physiology, including the immune response to infectious agents, which is mediated by the activation of the transcription factor NF-κB. Thus, understanding the mechanisms by which circadian clocks regulate NF-κB is a necessary step toward the development of improved therapies underwritten by NF-κB manipulation. Previous reports have identified OTUD7B a deubiquitinase, as a novel regulator of noncanonical NF-κB signaling, largely through the maintenance of TRAF3, a negative regulator of NF-κB. In investigations in our laboratory, when the Clock gene was repressed by shRNA, the results of microarray analysis demonstrated that Otud7b was down-regulated. Further research by real-time PCR and western blot revealed that that Otud7b exhibits rhythmic mRNA expression. These findings confirm Otud7b as a novel clock-regulated gene. Additionally, Otud7b may be an important bridge between the circadian system and noncanonical NF-κB pathway regulation.

Simulated microgravity influences circadian rhythm of NIH3T3 cells

Abstract Gravity heavily influences living organisms on earth including their circadian rhythm, which is fundamentally important for coordinately physiology in organisms as diverse as cyanobacteria, fungus and humans. Numerous researches have revealed that microgravity in outer space can affect circadian rhythm of astronauts and rodent animals, but the mechanism remains unknown. Using rotary cell culture system to simulate microgravity environment, we investigated the role of simulated microgravity in regulating the circadian rhythm of NIH3T3 cells. Our experiments found that simulated microgravity can not only influence the mRNA level of some core circadian genes, but also modify the circadian rhythm of Per1 and Per2 synchronized after phorbol myristate acetate treatment. Remarkably, MEK/ERK pathway was transiently activated after a 2-h simulated microgravity treatment, with a significant upregulation of Kras, Raf1 and p-ERK1/ERK2. Moreover, U0126, a selective inhibitor of MEK/ERK pathway, could disrupt the circadian rhythm of Per1 and Per2 synchronized after simulated microgravity treatment. Together, our results unveil that simulated microgravity could act like a zeitgeber to influence the circadian rhythm of NIH3T3 by acting on MEK/ERK pathway, indicating that MEK/ERK pathway may act as a bridge which connects cells mechanotransduction pathway and circadian rhythm regulation.