目的 近日节律系统作为生物体中最为重要的稳态调节系统在维持机体正常功能的过程中发挥着非常重要的作用。近日节律的破坏会导致机体罹患包括癌症在内的各种疾病，近日节律不仅影响着神经退行性疾病、心血管疾病等相关疾病的发生发展，且对这些疾病的治疗和预后有显著影响。基因小鼠模型和人类临床研究表明，大部分肿瘤细胞的近日钟基因在转录过程中存在基因突变。因此，了解近日节律系统在肿瘤发生发展中的作用，已经成为了时间生物学领域最为热门的研究课题。研究者们希望通过确定核心生物钟基因在肿瘤进程中发挥何种作用，以明确其影响肿瘤发生的具体调控机制，进一步评估节律基因作为关键靶点的潜在可能性，为抑制甚至是治疗肿瘤找出新方法。为了明确回答上述问题，探索节律系统对肿瘤的影响机制，本研究通过shRNA特异性沉默了核心节律基因Clock的表达，研究Clock基因在肿瘤细胞生长、转移中的作用，并尝试进一步探讨近日节律系统调控肿瘤发生的分子机制，以期为肿瘤的分子治疗提供新的思路。 方法 首先我们设计并构建了下调Clock表达的慢病毒载体，将其转染到小鼠乳腺癌4T1细胞株(Mouse mammary carcinoma cell)内，通过CCK8法绘制细胞增殖曲线，普通平板克隆、流式细胞周期分析、Transwell小室培养、Real-time RCR、Western blot等方法研究Clock低表达对体外培养的小鼠乳腺癌4T1细胞增殖和迁移的影响；接下来我们通过Balb/c雌鼠右前肢腋窝皮下成瘤模型和尾静脉注射建立肺转移模型来验证体外结果，检测Clock基因对4T1细胞在体内的增殖、迁移和侵袭能力的影响，进而检测上皮间充质转化(EMT)过程中几个标志性蛋白因子(如：E-cadherin、IQGAP1等)的表达水平变化。为了验证肿瘤迁移相关因子是否通过Clock基因而受到节律系统的调控，我们在体外通过马血清休克诱导技术使培养细胞内基因的节律表达实现同步化，在体内通过光暗循环诱导使Balb/c小鼠节律同步化，采用Real-time PCR和Western blot等技术手段检测候选基因表达是否具有节律特征，并确定是否为钟控基因；采用蛋白质免疫共沉淀技术检测候选基因是否和生物钟基因Clock在蛋白水平发生结合，以此来探寻节律系统在肿瘤发生发展中的作用。 结果 通过转染慢病毒载体成功构建了Clock低表达细胞株，CCK8检测细胞活力并绘制细胞增殖曲线，发现Clock被干扰后在一定程度上会促进乳腺癌细胞的增殖(p＜0.01)；平板克隆实验经计数后发现Clockt敲降后4T1形成的单克隆数也明显增加(p＜0.05)；采用碘化丙啶(PI)单染细胞核进行流式细胞周期分析检测发现Clock干扰后细胞的周期时相发生改变，处于G₀/G₁期的细胞比例有轻微的下降，而处于S期的细胞比例增多(p＜0.05)；在小鼠皮下成瘤实验中发现Clock低表达细胞株形成的瘤块体积比对照组4T1细胞株所形成的瘤块体积大(p＜0.01)；这些结果都表明Clock基因能够抑制小鼠乳腺癌细胞4T1的增殖。接下来通过Transwell小室培养实验发现干扰后穿膜细胞数与对照组相比明显增加(p＜0.01)，说明抑制内源性Clock基因表达可显著促进4T1细胞迁移；小鼠尾静脉注射实验也同样发现与对照组细胞相比Clock低表达细胞株4T1的迁移和侵袭能力都有较为明显的提高；体外马血清休克诱导细胞同步，体内光暗循环诱导Balb/c小鼠节律同步，发现迁移相关因子E-cadherin、IQGAP1、 vimentin的基因表达和蛋白水平变化具有明显的节律特征，证实它们都是受生物钟调控的基因即钟控基因；通过免疫共沉淀实验发现CLOCK和E-cadherin在蛋白水平上可以相互结合。 结论 Clock基因在小鼠乳腺癌细胞株4T1中低表达的体外实验和Balb/c小鼠皮下成瘤和尾静脉注射的体内实验都显示，Clock低表达可以明显地促进乳腺癌细胞4T1的生长和转移，E-cadherin、IQGAP1、vimentin的表达呈明显的节律特征，且CLOCK和E-cadherin在蛋白水平上可以相互结合，提示Clock可能会参与到上皮间充质转化(EMT)介导的肿瘤细胞的转移过程中并引起相关因子的表达发生变化。 关键词：近日节律；Clock基因；细胞增殖；细胞迁移；EMT；乳腺癌

 Objective Circadian rhythm plays a very important role in maintaining the normal function of the body as the most important stable regulation in organism. Disruption of the circadian rhythm may increase the risk of disease, including cancer. Circadian rhythm not only affects the development of neurodegenerative diseases, cardiovasculor diseases and other diseases, but also affects the treatment and prognsois of these diseases. Gene mouse models and human studies revealed that the circadian rhythm transcription mechanism in the cancer models have genetic mutations. Therefore, understanding the role of rhythm system in the development of tumors has become the most popular research in chronobiology. Researchers want to confirm the function of the core regulatory genes of circadian rhythm, then find out the specific regulation mechanismn of the occurrence of tumor, it may be possible to find out a new method which can suppress even treat cancer. In order to clearly answer these questions and explore the mechanism of circadian rhythm affecting tumors. In the present research, we applied shRNA to specifically silence the expression of Clock gene, then evaluated the role of the Clock gene in the proliferation and migration of mouse breast cancer cells 4T1, and investigated its possible regulatory pathways and mechanisms. This pathway, regulated by the Clock gene, provides a clearer picture between circadian clock disorder and breast cancer, and provides new ideas for tumors molecular therapy. Methods We designed and constructed adeno-associated lentivirus expressed by down-regulation of Clock and transfected 4T1 cells of mouse mammary carcinoma. Curves of cell proliferation were drew via CCK8 assay. We also detected extracorporeal influence of CLOCK on the proliferation and migration of breast cancer cells by normal plate clone, analysis of cell cycles of flow cytometry, transwell test, Real-time PCR and Western blot. In addition, we investigated the effects of low expression of Clock on tumor growth in mice. Subcutaneous tumor and tail vein injection model in Balb/c mice are established to detection Clock effects on cell proliferation, migration and invasion in vivo. We found that the expression of epithelial-mesenchymal transition related genes like E-cadherin and IQGAP1 changed. To verify whether the migration related factors are regulated by the circadian rhythm through Clock, the horse serum shock induction technique was used to synchronize the cells rhythms in vitro, the light-dark cycle induction technique was used to synchronize Balb/c mice rhythms in vivo. Real-time PCR and Western blot were used to detect whether the candidate gene expression was rhythmic; Detected whether the candidate gene was combined with Clock gene by co-immunoprecipitation and explored the role of rhythm system in the development of cancer. Results The Clock knock down cell line 4T1 were successfully established, after Clock was down regulated, we found proliferation vitality of breast cancer cells improved a little by the assay of CCK8 (p<0.01) , clone formation assay found the number of monoclonals were increased after Clock was down regulated (p<0.05) ; we detected cell cycle with FCM and found the number of cells of G0/G1 phase was decreasing but S phase was increasing (p<0.05) , which further explained the interfered Clock could facilitate the proliferation of cells in some degree; We also found tumor volumn was larger in the Clock knock down cell line 4T1 compared with control group (p<0.01) ; All these results indicate that Clock can inhibit the proliferation of cells to a certain degree. Transwell assay found that in the Clock knock down cell line 4T1 the number of transmembrane cells increased significantly compared with the control group (p<0.01). This, coupled with the observed increase in migration of the knocked down cells, illustrates an endogenous inhibitory role of Clock on cellular proliferation and migration; Mouse tail intravenous injection test found that Clock knock down cell line 4T1 migration and invasion have obvious inhibition compared with the control group; Horse serum shock induced cell synchronization in vitro, light-dark cycle induced Balb/c mice rhythms synchronization in vivo, Real-time PCR and Westem-blot showed that the gene expression and protein level of E-cadherin, IQGAP1 and vimentin had obvious rhythmic characteristics, and confirmed that they are clock-controlled genes. Coimmunoprecipitation assays showed that CLOCK is coimmunoprecipitated with E-cadherin. Conclusions Downregulation of Clock may promote the proliferation and migration of mouse breast cancer cells 4T1 in vitro and in vivo. The expression of E-cadherin, IQGAP1 and vimentin had obvious rhythmic characteristics, CLOCK and E-cadherin can bind to each other on protein level which indicate that the Clock participates in the metastasis of epithelial mesenchymal transformation (EMT) mediated tumor cells and causes changes in the expression of related factors. Keywords: Circdian rhythm; Clock; Cell proliferation; Cell migration; EMT; Breast cancer