目的 通过对睾丸精子超微结构的观察和实验室前期数据的生物信息学再分析,探讨睾丸Clock基因下调使小鼠精子前向运动百分率降低的机制.方法 选取SPF级ICR雄性小鼠(8周龄)14只,随机分为实验组和对照组,每组6只,另2只小鼠作为免疫共沉淀实验样本.获取小鼠Clock基因下调的睾丸组织,透射电镜观察其中精子的超微结构.结合对实验室前期数据再分析的结果,ELISA检测睾丸组织中α-KGDH的活性,并以RT-qPCR检测睾丸中二氢硫辛酸脱氢酶(DLD)的转录组水平.最后在野生型小鼠睾丸组织中验证CLOCK蛋白与DLD蛋白的相互作用关系.结果 与对照组相比,实验组Clock基因的表达显著降低(P＜0.001);实验组睾丸中精子细胞线粒体形态和排列出现异常;差异蛋白的GO和KEGG富集分析提示,实验组精子线粒体中三羧酸循环限速酶α-KGDH的亚基构成蛋白之一的DLD明显下调(P＜0.05);与对照组相比,实验组睾丸α-酮戊二酸脱氢酶复合体(α-KGDH)酶活性明显降低(P＜0.05);与对照组相比,实验组睾丸Dld基因mRNA水平也明显下调(P＜0.05);而野生型小鼠睾丸中CLOCK蛋白与DLD蛋白能够相互作用.结论 睾丸Clock基因的下调可导致小鼠精子线粒体结构和功能蛋白发生改变,从而限制了线粒体能量的产出,最终导致精子前向运动百分率降低.

The synchronization of the circadian signals to external or suprachiasmatic nucleus stimulation in the peripheral clocks is essential for maintaining the usual function of human body. However, aging will disrupt the synchronization of peripheral circadian rhythms, thus leading to some age-associated diseases. Up to now, little is known about the modification of the oscillatory rhythms in aged cells. A recent report showed that cell senescence in vascular human smooth muscle cells (HSMCs) altered circadian rhythms by a dysregulation of rhythmic gene expression. Furthermore, this alteration could be reversed by telomerase reconstitution. To test whether telomerase reconstitution can restore disrupted circadian rhythm in other types of senescent cells, we used fibroblasts as cell models to profoundly investigate the relationship between cell senescence and circadian rhythm modulation. We found that the response of rhythmic gene expression to serum stimulation was markedly attenuated in senescent fibroblasts, telomerase-reconstituted fibroblasts reset the circadian oscillation of rhythmic gene expression, and the activation of pERK-CREB and p38-CREB pathways might be involved in the circadian rhythm resetting. These findings suggested that telomerase reconstitution might be a good way to reset synchronization of peripheral circadian rhythms disrupted in senescent tissues.

Emerging evidence has demonstrated that long noncoding RNAs (lncRNAs) play critical roles in the epigenetic and transcriptional regulation of mammalian circadian systems. Circadian rhythmicity regulates many aspects of our immune system, and perturbation of the circadian clock can augment the inflammatory response. However, knowledge of the precise functions of lncRNAs in the regulation of immune functions within the circadian system is relatively limited. In this study, differentially expressed lncRNAs induced by Clock knockdown were screened via mRNA/lncRNA microarray and bioinformatic prediction analysis. We identified a Clock-regulated lncRNA, AK028245, which was correlated with the activation of the immune response. The expression levels of AK028245 were decreased in the spleen of immunosuppressed mice and elevated in immune-activated mice treated with lipopolysaccharide (LPS). Further, Clock knockdown decreased the expression of OTUD7B and A20, 2 early immune response factors acting on the NF-κB signaling pathway. Interestingly, inhibition of AK028245 increased their expression, mitigating the effects of Clock knockdown. In addition, inhibition of AK028245 downregulated the expression of tumor necrosis factor-α and interleukin-6 in the late stages of LPS stimulation and the expression of interferon-γ and Cxcl12 in the peak stages. We conclude that this newly identified lncRNA plays a role in the crosstalk between Clock and immune response regulators, likely resulting in a proinflammatory response targeting OTUD7B and A20. The lncRNA AK028245 has revealed a new mechanism of the immune response and provided new targets for the treatment of immune disorders.

Circadian rhythm has been involved in the regulation of many physiological activities. Autophagy is the metabolic process that transports substances in the cytoplasm to the lysosomes for degradation, and involved in the process of many diseases, including cancer. Studies have shown that autophagy activity has a circadian rhythm feature. As the core gene of the circadian rhythm system, clock has participated in the occurrence and development of cancer. The expression of clock whether regulates tumor development by autophagy has not been illustrated at present. In this study, we established a stableclock-knockdown strain of mouse breast cancer cell 4T1 to explore the changes in autophagy activity. The results showed that autophagy-related proteins ATG9A, ATG7, LC3B and Beclin1 (ATG6) were down-regulated withclock-knockdown, and the p62, the key factors of autophagy, was up-regulated. Moreover,clock-knockdown caused significant up-regulation of p65, and inhibition of I kappa B alpha and p-AKT?which were the core factors of several signaling pathways involved in autophagy. Serum rhythm-inducing experiments behaved that the expression of LC3B and Beclin1 had rhythmic characteristics?differed from LC3A. The regulative geneclockwas initially established to regulate the autophagy-related genes, and the suggested direction of its regulatory pathways.

Circadian rhythms help organisms adapt to changes of external environment by regulating energy metabolism and remaining the balance of homeostasis. Numerous researches have proved that the physiological function of liver was precisely controlled by circadian rhythms. Clock , one of core circadian genes, has been demonstrated to regulate the oxidative phosphorylation process of mitochondrial, which provides energy for living cells and acts as one of the hub for apoptosis. However, whether Clock gene regulates mitochondrial apoptosis pathways in liver cells remains less explored. In the present study, we used lentiviral vector to establish a stable AML12 cell lines which were capable of expressing specific shRNA to interfere the expression of Clock gene and investigated the effect of Clock on mitochondrial apoptosis pathways. Herein, we found that the interference of Clock gene could significantly suppress mitochondrial apoptosis pathways by stabilizing mitochondrial membrane potential and inhibiting mitochondria out membrane permeablization, which might be a result of lower expression of BAD and BIM proteins. Moreover, the interference of Clock gene could downregulate the expression of mitochondrial apoptosis factors, i.e. AIF, CYCS, APAF-1 and SMAC, which will suppress the formation of apoptosome and the process of DNA degradation to further inhibit apoptosis process. This work provides an insight on the important role of Clock gene participating in mitochondrial apoptosis pathways of hepatocytes and unveils a probable pathogenesis of how circadian rhythm regulates liver diseases.

Doxorubicin (DOX) is a potent anti-neoplastic agent with cumulative cardiotoxicity. DOX-induced cardiotoxicity has been shown to depend on the different dosing times. However, the basis for determining the dosing time to minimize DOX-induced cardiotoxicity and the underlying mechanisms remain incompletely understood. Here we first showed that SIRT3, the major mitochondrial deacetylase, is negatively correlated to DOX-induced cardiotoxicity through the regulation of ATP production, mitochondrial membrane potential (MMP) level and ROS level in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Then, we used in vivo experiments to demonstrate that DOX significantly reduced the SIRT3 expression and the SIRT3 activity as reflected by the increased AcK68MnSOD/MnSOD ratio in rats after six weeks of treatment. Notably, the activity of SIRT3 had an obvious diurnal rhythm pattern in the myocardium of healthy rats. More importantly, an obvious lower AcK68MnSOD/MnSOD ratio was observed in rat hearts with DOX administrated at Zeitgeber time (ZT) 9 (ZT 0 was the time lights were turned on) than ZT1, which represent the peak and trough of SIRT3 activity. Moreover, DOX ZT9 reduced the body weight loss, extended the survival period, improved the heart function and alleviated the myocardial lesions compared to DOX ZT1. Mechanistic investigations demonstrated that DOX ZT1 significantly reduced ATP production, oxygen consumption rate (OCR) at various respiration states, MMP level and MnSOD activity and enhanced the H2O2 level compared with CON ZT1, whereas there was no significant effect for DOX ZT9 compared with CON ZT9. Taken together, dosing at the peak time of SIRT3 activity reduced DOX-induced cardiotoxicity, which may be related to the increased endogenous tolerance against the mitochondrial dysfunction and oxidative stress caused by DOX.

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.

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.

Objective To study the effect on the DNA methylation level of MS-1 cell by inhibiting the expression of Clock gene,and explore the mechanism underlying Clock gene regulation of cell proliferation.Methods We knockeddown the expression of Clock gene by RNA interference,detected the DNA methylation level by Methylated DNA Quantification Kit,and the expression of Mecp2 and β-Catenin by Real-time PCR and Western blot.Results Knock-down the expression of Clock gene causes reduced DNA methylation level of MS-1 cell,and the expression of Mecp2 and β-Catenin.The DNA methylation levels of Clock gene Koncked-down group and Neagtive Control group(NC) were 1.067±0.034％ and 1.983±0.154％,p＜0.05,respectively.The Real-time PCR results display the expression of Mecp2 and β-Catenin in CK group were 0.98824 ± 0.12243 and 1.00000 ± 0.09547,compare with the NC group 1.80979 ± 0.18520 and 1.62584 ± 0.17167,respectively.The relative protein levels of MECP2/β-TUBLLIN and β-CATENIN/β-TUBLLIN were 0.289546± 0.104738 and 1.093368 ± 0.214433 in CK group,compare with 0.649544 ± 0.140909 and 3.305374 ± 0.260016 in the NC group,respectively.Conclusion The DNA methylation level of MS-1 cell could be affected by Clock gene.Clock gene may regulate cell proliferation by inhibiting the expression of Mecp2 and β-Catenin.

The circadian rhythm is one of the basic systems in an organism. It helps the organism maintain harmony with the daily changes of the external environment to ensure proper physiological activities. Previous studies from our laboratory have indicated that the miR-29a/b/c can bind to the circadian clock gene hPer1 at the 3 UTR region and regulate its mRNA and protein expression, affecting various organismal physiological processes. Meanwhile, it has been reported that the circadian gene Per plays a role in the regulation of the early growth response gene Egr2, which plays an important role during midbrain development. Here, we confirmed that miR-29a/b/c regulates Egr2 function through mPer1 binding, which elucidates a novel connection between mPer1 and Egr2.

Drug abuse is characterized by its long term persistence which has physical and psychological dependence. Clinical treatment, psychological counselling and social intervention can help patients have a certain withdrawal from physical dependence, but psychological addiction remains a serious problem leading over 90% of relapse. Influence of social interaction on drug effects has a close relationship with psychological dependence. In order to study the enduring changes in long-term memory in brains of drug-withdrawal mice, we established a social interaction model to compared hippocampal and prefrontal cortex gene expression in drug abuse mice. Two groups of physical morphine-withdrawal mice were caged in morphine abuse mice and saline-treated mice for four weeks, respectively. Morris' water maze was used for the detection of learning and memory, magnetic resonance spectroscopy (MRS) was used for the integrity of neurons in the brains between two groups. Expression GABA(A) and PKC alpha which mediated neurotransmission of GABA and glutamate in hippocampus and the prefrontal cortex were determined by immunohistochemistry and Western blot. The results showed that compared with morphine-withdrawal mice that social interacted with saline treated mice, the ones who lived with morphine abuse mice had a decreasing learning and memory ability and much more damaged neurons. Moreover, overexpression of mRNA and protein levels of GABA(A) and PKC alpha reveals the upregulation of GABA and downregulation of glutamate in neurons, enhancing the learning and memory in morphine withdrawal mice to trigger the reward pathway in morphine addiction at first. However, over activation of GABA and glutamate inside or outside of neurons had a negative feedback in learning and memory function. In conclusion, drug-withdrawal mice that social interacted with drug abuse mice had an increasing memory caused by environmental stimulus, but the over simulations may lead to neuron damages and a drop in functions of learning and memory through the over expression of GABA(A) and PKC alpha.

Objective To explore whether HIV TAT-protein can affect the expression of Clock,Cry,Bmal1,Per in the cell.Methods HIV-Tat protein expression plasmids were transfeeted into PC-12 with lipofectamineTM 2000,and Tat was expressed in PC-12.mRNA and protein were extracted 48 hours after transfection,and the expression of rhythm gene was detected by real-time quantitative PCR and Western-blot,respectively.Resnlts The results showed that the expression level of Cry1 was significantly increased(P＜0.05).The expression of Clock was significantly decreased(P＜0.05).The expression of Bmal1 and Per was not significantly changed in transfected Tat-expressing plasmid compared with untransfected and empty plasmid.Conclusion HIV Tat affects the expression of the circadian genes Cry1 and Clock,which is related to the decrease of Clock expression and the increase of Cry1 expression,Suggesting that TAT protein can affect the expression of rhythmic genes and may regulate the physiological function of cells through the influence of circadian rhythm.

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-kappa B. Thus, understanding the mechanisms by which circadian clocks regulate NF-kappa B is a necessary step toward the development of improved therapies underwritten by NF-kappa B manipulation. Previous reports have identified OTUD7B a deubiquitinase, as a novel regulator of noncanonical NF-kappa B signaling, largely through the maintenance of TRAF3, a negative regulator of NF-kappa B. 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-kappa B pathway regulation.

目的 探讨NIH3T3细胞中miR-29b对血清诱导的立早基因Egr2 mRNA表达的影响及其机制.方法 将体外培养NIH3T3细胞分为实验组、阴性对照组和空白对照组.实验组用miR-29b转染,阴性对照组空转,而空白对照组则不做任何处理.RT-qPCR检测近日节律基因mPer1与立早基因Egr2 mRNA的表达水平.结果 实验结果显示,mPer1和Egr2的表达均发生了较明显的变化.与阴性对照组相比,空白对照组中mPer1和Egr2的mRNA表达水平无明显差异;而实验组中mPer1的mRNA表达降低了42％,Egr2的mRNA表达升高了49％.结论 miR-29b能上调Egr2的表达,其途径可能是通过抑制NIH3T3细胞近日节律基因mPer1的表达实现.

目的 探究2000μW/cm2电磁辐射对小鼠运动节律和Clock基因表达水平的影响.方法 将4周龄的雄性昆明鼠30只随机分为三组:空白组,1h/d辐射组,2h/d辐射组.固定小鼠,使其接受2000μW/cm2电磁辐射,连续10天,之后监测其转轮活动的近日节律变化,并且提取小鼠脑组织中的总RNA和蛋白,RT-PCR和western-blot检测三组之间Clock基因和蛋白的表达是否存在显著性差异.结果 与空白组相比,两个辐射组小鼠近日节律均有缩短的现象(P＜0.05),且Clock基因和蛋白的表达水平显著性的升高(P＜0.05).结论 2000μW/cm2电磁辐射会缩短小鼠的近日节律周期,其机制可能与Clock基因表达升高有关.

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.

目的 探讨节律基因Clock对胶质瘤C6细胞增殖的影响及其机制.方法 通过小片段干扰RNA(smallinterfering,siRNA)特异性沉默C6细胞Clock基因的表达.采用CCK-8试剂盒检测C6细胞的增殖情况,并利用流式细胞仪检测Clock基因沉默后C6细胞的细胞周期分布,通过荧光定量Q-PCR检测β-catenin和Tcf1 mRNA的表达水平.结果 与空白对照组和阴性对照组相比,SiClock转染组C6细胞增殖活力明显降低,S期细胞所占比例明显下降,同时β-catenin和Tcf1 mRNA的表达也明显降低(均P＜0.05).结论 干扰节律基因Clock表达,能够抑制胶质瘤细胞的生长.其机制可能是通过Wnt/β-catenin信号通路调节其作用.

The present paper reports the effect of pancreatitis induced by cholecystokinin (CCK) on free-running rhythm of locomotor activity of the ICR mice, and analyzes the interaction of inflammatory diseases and acute pancreatitis with circadian rhythm system. In the study, the mice were modeled under different phases of acute pancreatitis in DD status (Double Dark, constant dark condition). By comparing of the inflammatory status and the indicators of rhythm before and after modeling of the running wheel activity group and the rest group, it was observed that the rest group showed more possibility of inflammation than the activity group did in ICR mice model of acute pancreatitis. In the rest phase model, the extension of the period is particularly longer. The results presented indicated that CCK-induced acute pancreatitis impacted free activity rhythm of ICR mice. Also in a free running model under different phase, the inflammation severity was proved significantly different. This study provides possible clues for the research of the pathogenesis of acute pancreatitis severe tendency.

目的 探讨模拟微重力对NIH3T3细胞近日节律基因Per1,Per2与Cry1 mRNA表达的影响.方法 体外培养培养NIH3T3细胞分为实验组和对照组.实验组采用RCCS模拟微重力环境培养NIH3T3细胞;而对照组在正常重力环境下静置培养.两组均采用PMA刺激细胞,RT-qPCR检测Per1,Per2与Cry1 mRNA的表达水平.结果 对照组与实验组的近日节律基因Per1,Per2与Cry1的mRNA都呈现节律性.与对照相比,实验组节律基因的振幅减小,相位延迟.结论 模拟微重力影响NIH3T3细胞近日节律基因Per1,Per2,Cry1的表达.

BACKGROUND:Isorhamnetin (Iso), a novel and essential monomer derived from total flavones of Hippophae rhamnoides that has long been used as a traditional Chinese medicine for angina pectoris and acute myocardial infarction, has also shown a spectrum of antitumor activity. However, little is known about the mechanisms of action Iso on cancer cells.OBJECTIVES:To investigate the effects of Iso on A549 lung cancer cells and underlying mechanisms.MATERIALS AND METHODS:A549 cells were treated with 10~320 μg/ml Iso. Their morphological and cellular characteristics were assessed by light and electronic microscopy. Growth inhibition was analyzed by MTT, clonogenic and growth curve assays. Apoptotic characteristics of cells were determined by flow cytometry (FCM), DNA fragmentation, single cell gel electrophoresis (comet) assay, immunocytochemistry and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) . Tumor models were setup by transplanting Lewis lung carcinoma cells into C57BL/6 mice, and the weights and sizes of tumors were measured.RESULTS:Iso markedly inhibited the growth of A549 cells with induction of apoptotic changes. Iso at 20 μg/ml, could induce A549 cell apoptosis, up-regulate the expression of apoptosis genes Bax, Caspase-3 and P53, and down-regulate the expression of Bcl-2, cyclinD1 and PCNA protein. The tumors in tumor-bearing mice treated with Iso were significantly smaller than in the control group. The results of apoptosis-related genes, PCNA, cyclinD1 and other protein expression levels of transplanted Lewis cells were the same as those of A549 cells in vitro.CONCLUSIONS:Iso, a natural single compound isolated from total flavones, has antiproliferative activity against lung cancer in vitro and in vivo. Its mechanisms of action may involve apoptosis of cells induced by down-regulation of oncogenes and up-regulation of apoptotic genes.