【作者】 陈川；

【导师】 王阁；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2011， 博士

【摘要】 人类细胞中BTG/Tob蛋白家族的六个成员(BTG1,BTG2^PC3/Tis21,BTG3/ANA,BTG4/PC3B,Tob1/Tob和Tob2),在细胞周期调控、细胞生长与分化、DNA损伤修复等方面发挥着重要功能[1-5]。在多种肿瘤如肝癌、乳腺癌、肾癌中,BTG/Tob蛋白表达均降低或缺失,基因敲除小鼠肿瘤发生率明显高于对照组小鼠,表明BTG/Tob蛋白表达下调是肿瘤发生发展过程中的重要分子事件,其作为新的抑癌基因的功能正逐步得到证实。最新研究表明,BTG/Tob还参与细胞核内转录及胞浆mRNA脱腺苷酸化的调控,而与CCR4-NOT蛋白复合体的相互作用是BTG/Tob发挥这一功能的前提条件[6-8]。进化高度保守的CCR4-NOT蛋白复合体具有3’-5’核酸外切酶活性,在人类中包含9个亚基:CNOT1,CNOT2,CNOT3,CNOT6/hCcr4a,CNOT6L/hCcr4b,CNOT7/hCaf1,CNOT8/hPop2,CNOT9和CNOT10。其中,CNOT6,CNOT6L,CNOT7,CNOT8是细胞中主要的腺苷酸酶,通过脱腺苷酸化作用降解mRNA及特定转录产物[10-12],从而在真核生物的基因表达调控中发挥重要作用。研究发现除BTG4外,所有BTG/Tob家族蛋白均可与高度同源的CNOT7和/或CNOT8相互结合[13-17]。晶体结构研究揭示,由5个α螺旋结构、4个β折叠构成的BTG/Tob区域是BTG/Tob蛋白与CNOT7结合的特征性区域[18-20],而BTG2及Tob1 BoxA和BoxB区域的关键亚基也与CNOT7有相互作用[21],此外,最近还报道Tob1的C端区域也可与CNOT1亚基结合[22]。因此,BTG2及Tob1可通过相互结合作用招募CCR4-NOT复合体促进特定mRNA的脱腺苷酸化以发挥调控转录的功能。更重要的是,BTG/Tob与CNOT7结合复合体结构的形成对BTG/Tob蛋白有效发挥其抗增殖特性同样是必不可少的。这些研究高度提示BTG/Tob蛋白与CCR4-NOT蛋白复合体关系密切,二者相互结合,相互调控,共同参与多种细胞生物学行为的调节。前期研究发现,CCR4-NOT蛋白复合体在DNA损伤修复、组蛋白甲基化、精子发生等方面均发挥着重要作用,表明除参与mRNA降解之外,CCR4-NOT蛋白复合体各亚基还具有更重要的生物学功能。本研究在前期工作基础上,对CCR4-NOT蛋白复合体在神经系统中的新功能进行初步探索和揭示,旨在补充和完善其功能研究,为进一步阐明CCR4-NOT蛋白复合体的生物学意义提供实验依据。研究目的1.广泛研究CCR4-NOT蛋白复合体在小鼠脑组织及神经细胞中的表达模式,初步揭示其在神经系统中的新功能;2.探讨CCR4-NOT蛋白复合体抑制神经母细胞瘤细胞Neuro-2a分化的作用及可能分子机制。研究内容和方法1. BTG2及CCR4-NOT蛋白复合体在正常小鼠中的组织表达模式:利用Northern Blot和Western Blot方法广泛研究BTG2和CCR4-NOT蛋白复合体各亚基在正常小鼠中的组织表达情况,为进一步揭示其功能提供实验基础;2. CCR4-NOT蛋白复合体在小鼠脑组织及神经细胞中的表达模式:利用Western Blot和β-Gal Staining方法研究CCR4-NOT蛋白复合体各亚基在正常小鼠脑组织中的具体表达情况;培养神经干细胞,少突胶质细胞祖细胞等重要的神经细胞,通过维甲酸、去细胞因子等方法有效诱导其定向分化,利用Western Blot、Immunostaining、siRNA干扰等研究CCR4-NOT蛋白复合体各亚基在小鼠神经细胞中的表达模式及可能发挥的功能。3. CCR4-NOT蛋白复合体在神经母细胞瘤细胞Neuro-2a中的抑制分化作用:利用Neuro-2a作为神经样细胞分化模型,通过构建siRNA腺病毒载体和表达载体,研究在抑制和过表达CCR4-NOT亚基后对Neuro-2a分化的作用及可能分子机制。研究结果1. BTG2及CCR4-NOT蛋白复合体在正常小鼠中的组织表达模式: BTG2基因在正常大鼠肾脏中有较高丰度表达,在肺脏、肝脏、大脑、心脏呈低丰度表达,肌肉、胸腺、脾脏中无表达;CCR4-NOT复合体在小鼠各组织中呈广泛表达,尤其在大脑、肺脏、小肠、睾丸、胸腺、卵巢及脾脏中呈高表达。2. CCR4-NOT蛋白复合体在小鼠脑组织及神经细胞中的表达模式:CCR4-NOT蛋白复合体主要表达于大脑发育早期及端脑组织,在神经系统的三种主要细胞星形胶质细胞、神经元、少突胶质细胞中均有表达,各亚基间表达模式并不完全相同,部分亚基在神经干细胞中的表达高于在原代培养神经元中的表达,并随神经干细胞分化表达降低。CCR4-NOT复合体在少突胶质细胞祖细胞和少突胶质细胞中表达水平基本一致,但抑制其表达后少突胶质细胞增殖明显受到抑制。3. CCR4-NOT蛋白复合体在神经母细胞瘤细胞Neuro-2a中的抑制分化作用: CNOT6,CNOT8亚基在Neuro-2a细胞分化过程中表达逐渐降低,抑制其表达后伸出神经突的Neuro-2a细胞比例,神经突的数量、长度都较对照组明显增加,而过表达CNOT6,CNOT8能部分逆转CNOT6,CNOT8“敲除”细胞的表型。抑制CNOT6,CNOT8后,β-catenin的表达高于对照组细胞。结论1. CCR4-NOT蛋白复合体表达的时空特异性高度提示其可能是调控神经细胞增殖和分化的重要分子之一,进一步揭示了CCR4-NOT复合体的新功能。2. CNOT2,CNOT3,CNOT6,CNOT8,CNOT9亚基在神经干细胞中的表达高于原代培养神经元,并随神经干细胞的分化表达逐渐降低,提示这些亚基在神经干细胞发育和分化中可能发挥重要作用。3. CCR4-NOT蛋白复合体不参与少突胶质细胞祖细胞向少突胶质细胞的分化过程,但在维持细胞增殖和存活中发挥重要作用。4.抑制CNOT6或CNOT8表达可促进Neuro-2a细胞神经突的生长及延长,而过表达能部分逆转“敲除”细胞表型,其分子机制可能是通过抑制β-catenin等促神经突生长的转录因子活性。更多还原

【Abstract】 The mammalian BTG/Tob family comprises six proteins (BTG1, BTG2^PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob and Tob2) in human cells, which regulate cell cycle progression, cell growth and differentiation, DNA damage repair,tumourigenesis and cancer suppression in a variety of cell types. Down regulation or impaired expression of BTG/Tob is observed in several tumors, including prostate cancer, clear renal cell carcinomas and breast cancer, mice lacking BTG/Tob are prone to spontaneous carcinogenesis with high frequencies of tumors found in liver, lung and lymph nodes, indicating the deregulation of BTG/Tob early at the tumorigenic process may also be an important step in the development of mammary tumors and BTG/Tob as new tumor suppressor is demonstrated by more and more evidence.Several members of the BTG/Tob family is shown to be implicated in transcription in the nucleus and cytoplasmic mRNA deadenylation and turnover in recent studies, moreover, BTG/Tob factors impact deadenylases through the interaction with DNA-binding transcription factors as well as the paralogues CNOT7 （human Caf1/Caf1a） and CNOT8 （human Pop2/Calif/Caf1b）, two deadenylase subunits of the CCR4-NOT complex.The CCR4–NOT complex conserved from yeast to human is composed of nine subunits, CNOT1, CNOT2, CNOT3, CNOT6/hCcr4a, CNOT6L/hCcr4b, CNOT7/hCaf1, CNOT8/hPop2, CNOT9 and CNOT10, among which CNOT6, 6L, 7, 8 possess exonuclease domain, and are implicated in several aspects of mRNA metabolism, and that both factors have a predominant role in mRNA poly（A） tail shortening in vivo.Except for BTG4, all BTG/Tob proteins can interact with the highly similar paralogues CNOT7 and/or CNOT8,whose amino acid sequences are 76% identical （89% similar）.Studies on crystal structures of the conserved BTG/Tob domain of BTG2 and Tob1 revealed that it is composed of fiveα-helices and fourβ-strands, and this characteristic structure contacts with CNOT7 surface.Moreover, key residues from both box A and box B motifs of Tob1 are involved in the Tob1–CNOT7 interaction, an interaction between Tob1 and the CNOT1 subunit of the large CCR4–NOT assembly was also described recently. These observations suggested that BTG/Tob proteins might direct CNOT7 to its mRNA substrates in controlling mRNA deadenylation. More importantly, the association between Tob and CNOT7 appears to be crucial for the exertion of a strong anti-proliferative activity.We previously showed that depletion of CNOT6L or CNOT7 subunit of the complex impaired cell growth or spermatogenesis in mice, respectively, suggesting that the CCR4-NOT complex has non-housekeeping functions despite its fundamental cellular function in mRNA metabolism. However, the functions and underlying mechanism of action of most subunits remain largely unknown. To analyze their biological significance, we performed extensive analysis of their expression in mouse central nervous system to establish the foundation for further study of CCR4-NOT complex.Objective1 Study extensively on the expression of CCR4-NOT complex in mouse brain tissues and different cell types, and to reveal its new functions in central nervous system.2 Study the functions and underlying mechanisms of CCR4-NOT complex on cell differentiation by applying the Neuro-2a mouse neuroblastoma cell line as model system.Materials and Methods1 Expression of BTG2 and CCR4-NOT complex in various mouse organs: Northern Blot and Western Blot were used for analysis of expression pattern.2 Expression of CCR4-NOT complex in different regions and cells of brain: Western Blot andβ-Gal Staining were used for analysis of expression pattern. Primary culture of neural stem cells （NSCs） and oligodendrocyte progenitor cells （OPCs） were induced by retinoic acid （RA） and removal of cytokines. Western Blot、Immunostaining、siRNA were applied for study on the expression and functions of CCR4-NOT complex.3 The functions of CCR4-NOT complex in Neuro-2a cells: The retrovirus vectors pSIREN-CNOT6 , pSIREN-CNOT8 for knockdown and pMX-EGFP-CNOT6 ,pMX-EGFP-CNOT8 for over expression were constructed. The effect of CCR4-NOT complex on Neuro-2a differentiation was studied after CNOT6 or CNOT8 knockdown and over expression. Results1 Expression of BTG2 and CCR4-NOT complex in various mouse organs: BTG2 was expressed abundantly in kidney, lowly in the lung, brain, liver and heart, not expressed in muscle, thymus and spleen.CCR4-NOT complex subunits were expressed in most of mouse tissues, being especially high in the brain, lung, small intestine, testis, thymus, ovary, and spleen.2 Expression of CCR4-NOT complex in different regions and cells of brain: We further found that all CCR4-NOT subunits were highly expressed in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of mouse brains, moreover, they were highly expressed at early developmental stages. The brain consists of three major types of cells: astrocytes, neurons, and oligodendrocytes. All CCR4-NOT subunits were expressed in these three cell types, being particularly high in oligodendrocytes. The expression of several subunits of the CCR4-NOT complex were down-regulated in primary cultured neurons compared with in neural stem cells. The expression of CCR4-NOT complex were the same in OPCs and oligodendrycytes（OLs）, but cell proliferation of OLs were inhibited after CCR4-NOT complex knock down.3 The functions of CCR4-NOT complex in Neuro-2a cells: CONT6 or CNOT8 deletion promotes neurite outgrowth of Neuro-2a, on contrast, CONT6 or CNOT8 over expression inhibits neurite outgrowth, furthermore, over expression rescues the phenotype of CONT6 or CNOT8 knock down Neuro-2a cells,β-catenin was also unregulated in CONT6 or CNOT8 knock down cells.Conclusions1 CCR4–NOT complex may be a pivotal factor involved in the regulation of cell proliferation and differentiation, demonstrated by the expression pattern in the mouse brain.2 CNOT2, CNOT3, CNOT6, CNOT8, CNOT9 were down-regulated in primary cultured neurons compared with in NSCs, indicating CCR4–NOT complex may be required for maintaining the undifferentiated state of neural stem cells and involved in early neuronal differentiation or proliferation of NSCs.3 CCR4–NOT complex was not required in the differentiation of OPCs to OLs, but it play important role in regulating the proliferation of OLs.4 The inhibitory effect of CCR4–NOT complex on neurite outgrowth of Neuro-2a indicates that it may function during neuronal maturation throughβ-catenin inactivation.更多还原