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.

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.

Our previous study indicated that Clock gene could affect the thrombotic potential. In this present study, we examined the differential expression of proteins in Clock knockdown mice's plasma, including alpha 1-antitrypsin as a potential target. The proteins were examined in AML12 cells with Clock gene being knocked down to confirm the differential expressions. RNAs of those cells were extracted every 3 h in 24 h. The transcriptional levels of alpha 1-antitrypsin (Serpina1a) and fibronectin (Fn1) were analyzed with the least-squares fit of a 24-h cosine function by single cosinor method, but no circadian rhythm was determined in neither of these genes. The expressions of alpha 1-antitrypsin and fibronectin in Clock knockdown cells were found upregulated in both transcriptional and translational levels. Then, we applied ELISA assay to detect the concentration of activated protein C in Clock knockdown plasma and found a risen concentration. Fibronectin was reported to play a role in inhibiting the platelet aggregation, while activated protein C was demonstrated to inhibit PAI-1. All these results indicated that downregulation of the Clock gene in the circulatory system might have effects on PAI-1 by upregulating alpha 1-antitrypsin, and might play some roles in platelet aggregation by increased fibronectin.

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.

Objective: The aim of this study was to investigate the effects of IL-17a on neutrophil apoptosis in septic rats and its mechanisms. Methods: A total of 54 healthy adult male Wistar rats were randomly divided into 9 groups: normal control group (N), sepsis 12 hours and 24 hours group (S12 hour, S24 hour, respectively), sepsis + saline group (S12 hour+NS, S24 hour+NS, respectively), and low and high doses of recombinant IL-17 treatment 12 hours and 24 hours (S12 hour+L, S24 hour+L, S12 hour+H, S24 hour+H, respectively). Rats in the normal control group were injected with the same amount of normal saline and rats in each group were treated with intraperitoneal injections. Low and high doses of recombinant IL-17 were injected into the tail veins of each group of rats with rIL-17a 0.001 mu g and 1 mu g, while rats in each group were injected with an equal amount of normal saline. Rats were collected from the carotid artery, then the neutrophils were extracted by gradient centrifugation. Neutrophil apoptosis was detected by AnnexinV-FITC and PI double staining. Caspase 3 enzyme activity in neutrophils was detected by spectrophotography. Expression levels of Bax and Bcl-2 were detected by western blot. Changes in inflammatory factor IL-17a levels in the serum were detected by ELISA method. Results: Compared to the normal control group, neutrophil apoptosis rate was lower in the sepsis group than the control group (P<0.05). PMN apoptosis was significantly inhibited in rats of the sepsis group induced by rIL-17 tail vein injections at the same time point (P<0.05). Caspase-3 activity of 12 hours and 24 hours in the IL-17 high dose group was significantly higher than that in all other groups (P<0.05). In the IL-17 low dose group, the activity of caspase-3 in 12 hours and 24 hours was lower than that in other groups (P<0.05). Compared with levels of S-Fas in each group, in low dose IL-17 groups, either in 12 hours or 24 hours, expression levels of S-Fas were significantly higher than those in other groups (P<0.05), while in the high dose IL-17 groups, expression levels of S-Fas in 12 hours and 24 hours were lower than those in other groups (P<0.05). In addition, in high dose of IL-17 groups, levels of S-Fas in 12 hours and 24 hours were lower than those in other groups (P<0.05). In PMN of high dose of IL-17, expression of protein Bax was significantly higher than that in other groups (P<0.05) at 12 and 24 hours. Expression of BAX in low dose group of IL-17 on 12 and 24 hours was lower (P<0.05), while higher in the low dose group (P<0.05) than other groups. Conclusion: High doses of IL-17a promote activation of caspase 3 and expression of s-Fas, causing apoptosis of PMN, providing fundamental leads for the clinical treatment of sepsis.

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.

Traumatic brain injury (TBI) generally influences circadian rhythms and has been implicated changes in circadian rhythm. Whether TBI-induced changes in circadian rhythm may affect the prognosis or recovery from TBI remains to be investigated. Sixty-two patients with TBI were continuously monitored for intracranial pressure (ICP) during the first 24 hours after the implantation of ICP monitor. The data from each patient were analyzed using the least squares fit of a 24-h cosine function by single cosinor method. Parameters of circadian A (Amplitude)/M (MESOR) were used to evaluate the circadian rhythm of the patients. Student's t-test and Pearson's chi-squared test were utilized to analyze the differences between good prognosis group and poor prognosis. A linear regression analysis was then applied to calculate the correlation between circadian A/M of ICP and Glasgow Coma Scale (GCS) before discharge, the Extended Glasgow Outcome Scale (GOS-E), the dosage of mannitol, and time spent in the intensive care unit (ICU), respectively. The results demonstrated that circadian A/M of patients' ICP exhibited a positive correlation with GCS scores taken before discharge, GOS-E scores, and was negatively correlated with the amount of mannitol, and time spent in the ICU. We conclude that changes in the ICP circadian rhythm in TBI patients could reflect an internal signal of brain damage and, therefore, may be useful to predict a patient's prognosis and recovery from TBI.

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.

生物节律系统作为生物体的基本系统之一在稳态维持、新陈代谢、睡眠与觉醒和癌症的发生与治疗等生理及病理领域发挥了重要影响.而针对节律调节机制本身的研究也是生物研究的热点领域之一.作为生物研究的“新大陆”,非编码RNA (ncRNA)与节律系统和核心节律基因的相互作用日益为人所重视,已成为时间生物学领域最具潜力的研究课题之一.将综合国内外对非编码RNA与生物钟相互作用的研究现状,对参与其中的非编码RNA成员和机制做逐一综述.

Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements.

目的 通过干扰小鼠二细胞受精卵中节律基因Clock的表达,研究Clock对小鼠早期胚胎DNA甲基化的影响.方法 将Clock基因的RNAi干扰质粒转染到通过人工授精得到的二细胞受精卵中,通过移植假孕雌鼠获得7.5～11.5dpc的小鼠胚胎,并检测其基因组DNA甲基化水平和甲基转移酶Dnmt3a和Dnmt3b的表达.结果 Clock干扰组无论是基因组DNA甲基化水平还是甲基转移酶Dnmt3a、Dnmt3b的表达量均较对照组高(均P＜0.01).结论 降低小鼠受精卵中节律基因Clock的表达量,可以通过影响甲基转移酶Dnmt3a和Dnmt3b而改变其胚胎发育中DNA甲基化水平.

易普利姆玛(Ipilimumab)是一种特异性人细胞毒性T淋巴细胞抗原4(Cytotoxic T lymphocyte antigen 4,CTLA-4)抑制剂,美国食品药品管理局在2011年批准其专用于恶性黑色素瘤的肿瘤免疫治疗.近年来研究发现易普利姆玛对肺癌亦有良好疗效,联合放疗或其它药物治疗肺癌时效果突出,但同时也可能伴随一些不良反应.本文将根据最新研究对易普利姆玛治疗的基本原理、临床试验以及不良反应作一综述.