神经元限制性沉默因子对CART基因转录的调控作用及其机制研究
发布时间：2018-09-03 06:08
【摘要】：干细胞是一类具有极强自我更新及多向分化潜能的细胞,在体外特定条件下可被诱导分化为成熟的神经元、胰岛细胞,从而为细胞移植治疗糖尿病、帕金森氏综合症、中风等多种疾病提供优良的种子细胞,具有重要的临床应用价值。然而在干细胞向神经元、胰岛细胞分化的过程中存在着一系列问题,包括分化效率低、分化的细胞功能不成熟等,归根结底是因为分化的机制目前尚不清楚。文献调研及我们实验室前期的工作都表明神经元限制性沉默因子(neuron-restrictive silencer factor,NRSF;又称RE-1 silencing transcription factor,REST)在干细胞向神经元和胰岛细胞分化的过程中发挥重要的作用,此外,本实验室的研究还表明NRSF调控神经及胰岛相关的靶基因如胰岛素、可卡因-苯丙胺调节转录肽(Cocaine and amphetamine-regulated transcript peptide,CART)等的表达。基于这样的工作基础,进一步研究NRSF对其靶基因的转录调控机制,有助于我们更好的了解NRSF在干细胞向神经元或胰岛细胞分化及成熟细胞功能维持方面的作用,从而为今后指导干细胞向成熟的神经元或胰岛细胞分化提供新的思路。 NRSF是一种含有9个Cys2/His2型锌指结构的蛋白,具有一个DNA结合结构域、一个赖氨酸富含区、一个脯氨酸富含区和两个转录抑制结构域——N端抑制结构域和C端抑制结构域,广泛表达于胚胎干细胞(embryonic stem cells,ES细胞)、神经干细胞(neural stem cells,NSCs)和非神经细胞中,在大部分分化的神经元中没有表达,通过与特异的作用元件NRSE(neuron-restrictive silencer element,NRSE;又称Repressor Element,RE1)相结合,招募不同的抑制复合物,发挥对靶基因如II型钠离子通道、SCG10、连接蛋白36(Connexin36)等转录水平的调控,从而在维持ES细胞全能性与自我更新、胚胎早期发育、干细胞向神经元、胰岛细胞的分化等多个过程中扮演重要角色。 CART是一种新型的神经肽类物质,分布于中枢和周围神经系统及肾上腺、胰岛等内分泌组织,众多研究表明其参与到进食与肥胖、应激、能量代谢、神经保护等多个生理过程。近年来,有研究表明CART参与调控胰岛细胞的糖反应性。目前对CART的研究主要集中在CART肽的定位、加工及其功能发挥,关于CART基因转录调控方面包括转录因子、调控元件及CART肽发挥神经保护作用的机制的研究尚为数不多,而这些科学问题的探讨,可以使我们弄清如何使CART发挥生物学功能的分子机制,为基础研究和糖尿病、神经系统疾病等的临床治疗提供有力的理论指导。 我们之前的研究工作表明CART基因是NRSF作用的靶基因之一,其核心启动子区存在的NRSE样基序与NRSF的序列特异性结合可以发挥对CART基因的转录抑制调控。本文通过进一步的生物信息学分析发现,CART基因第一个内含子区也存在与公认的NRSE共有序列极为相似的NRSE样基序,并且在物种间具有较好的保守性,那么这段序列是否也参与了CART基因的转录调控?它又是如何与CART基因启动子区的NRSE相互协调发挥对CART的转录调节作用?目前关于CART基因转录调控机制方面的研究比较清楚的只有cAMP/PKA/CREB正性调控通路,那么,我们研究发现的NRSF-NRSE负性调控系统与cAMP/PKA/CREB正性调控系统之间是如何竞争性调节CART基因,共同实现对CART基因转录水平的调控呢?以上关键问题的解决也就成为了本研究的目的。 本研究主要包括三部分内容: 一、内含子序列对CART基因转录调控作用的研究 通过应用rVista软件,我们对从美国国家生物医学信息中心NCBI数据库检索得到的人、大鼠和小鼠CART基因的内含子序列进行分析,预测到了一系列可能调控CART基因表达的元件,包括ZF5、E47、NRSE、AREB6等,结合之前的工作基础,我们确定以人、大鼠、小鼠CART基因第一个内含子区保守性较好的NRSE样基序为研究对象,进一步开展CART基因转录调控机制方面的研究。为了探讨CART基因第一个内含子及其中的NRSE元件对CART基因的转录调控作用,我们基于CART启动子-荧光素酶报告载体pGL3-Basic-CART32(P-Luc),构建了荧光素酶基因3’端连入内含子序列(去或不去除NRSE基序)的荧光素酶报告载体pGL3-Basic-CART32-Intron(P-Luc-I)、pGL3-Basic-CART32-NRSEnon Intron(P-Luc-tI),将上述载体与含海肾荧光素酶的内参质粒pRL-CMV共转染入NTera2/CloneD1细胞,通过检测荧光素酶活性探讨CART基因第一个内含子区域及NRSE样基序对其转录活性的影响,结果显示P-Luc-I转录活性相对于对照组P-Luc下调57.9%(P0.05),表明CART基因第一个内含子序列参与CART基因的转录负调控;P-Luc-tI相对于P-Luc-I荧光素酶活性上调53.8%(P0.05),提示CART基因内含子通过其区域内的NRSE发挥对CART基因的转录负调控作用。之后我们通过应用anti-NRSF抗体进行染色质免疫共沉淀( chromatin immunoprecipitation,ChIP)实验,分析CART基因的内含子区域与NRSF的结合状况,结果显示二者之间存在直接的结合,证实NRSF与内含子区NRSE的结合真实的存在于天然染色质区。 二、NRSF对CART基因转录的调控作用及其机制研究 结合之前我们实验室对CART基因启动子区NRSE元件研究的工作基础及本文第一部分对CART基因第一个内含子区NRSE样基序的初步研究,为进一步探讨NRSF是如何通过CART基因启动子及内含子序列发挥对该基因的转录调控作用,我们开展了一系列的工作。通过电泳迁移率变动分析实验(Eelectrophoretic Mobility Shift Assay,EMSA)证实CART基因启动子区及第一个内含子区NRSE基序在体外均可以与NRSF发生序列特异性结合;染色质免疫共沉淀实验表明在NRSF表达量不同的细胞系,NRSF与NRSE结合后,通过招募组蛋白以不同的强度结合,形成不同的抑制复合物,进而发挥对CART基因的转录调控作用;为弄清NRSF是如何通过启动子及内含子序列发挥对CART基因的转录调控作用,我们使用了CART启动子-荧光素酶报告载体P-Luc、P-Luc-tI、P-Luc-I,同时构建了去除NRSE基序的CART启动子-荧光素酶报告载体pGL3-Basic-NRSEnon promoter (tP-Luc),及在其荧光素酶基因3’端连入内含子序列(去或不去除NRSE基序)的荧光素酶报告载体pGL3-Basic-NRSEnon promoter-Intron ( tP-Luc-I )、pGL3-Basic-NRSEnon promoter-NRSEnon Intron(tP-Luc-tI),将这些报告载体分别转染到HeLa细胞中,通过检测荧光素酶活性的分析结果表明CART基因内含子与启动子协同的参与CART基因的转录负调控,启动子区NRSF-NRSE对CART基因发挥更强的转录负调控作用。共转染荧光素酶报告载体、NRSF表达质粒pcDNA3.1-NRSF到NRSF低表达的SK-N-SH细胞中,也得到相似的结论;为了验证CART基因启动子与第一个内含子发挥的转录负调控作用是否依赖于其各自区域内的NRSE元件,我们在本室原有的含有双拷贝CART启动子NRSE基序(正常或突变序列)的报告载体的基础上,在其荧光素酶基因的3’端连入双拷贝CART内含子NRSE基序(正常或突变序列),将这些荧光素酶报告载体分别与含海肾荧光素酶的内参质粒pRL-CMV共转染入HeLa细胞和SK-N-SH细胞,检测荧光素酶活性的分析结果证实CART基因启动子及第一个内含子区对CART基因转录发挥的负调控作用依赖于其各自区域内NRSE元件的协同作用。 三、CART基因表达的正性与负性调控系统及其作用机制的研究 为了进一步考察NRSF-NRSE负性调控系统与cAMP/PKA/CREB正性调控系统之间是如何竞争性调节CART基因的表达,首先我们共转染P-Luc-I、对照质粒pcDNA3.1或呈浓度梯度的NRSF表达质粒pcDNA3.1-NRSF(100ng、200ng、400ng/2×105个细胞),检测荧光素酶活性的分析结果表明转染pcDNA3.1-NRSF 400ng/2×105个细胞可以抑制Forskolin/cAMP/PKA/CREB对CART的正性调控,提示高水平的NRSF表达引起的NRSF-NRSE负性调控作用可以抑制cAMP/PKA/CREB正性调控系统功能的发挥。 在此基础上,我们建立了神经细胞的氧糖剥夺(oxygen-glucose deprivation,OGD)模型,结合CART肽可以减轻缺血/缺氧导致的细胞死亡的功能开展了相关实验。近年来有研究表明局部脑缺血的大鼠或OGD处理的皮质神经元NRSF的表达出现上调,利用此模型我们可以进一步研究NRSF表达的变化是否会引起CART基因表达的变化,并影响cAMP/PKA/CREB正性调控通路的作用。 我们将接种在12孔板内的SK-N-SH细胞放置在37°C,0.3% O2,95% N2的低氧手套箱中处理3h,体外模拟缺血/缺氧的环境。通过RT-PCR和Western Blot检测处理的SK-N-SH细胞复氧24 h、48 h后NRSF、CART表达的变化,并应用流式细胞仪检测处理前后细胞凋亡率的变化,结果显示经OGD处理的SK-N-SH细胞复氧后NRSF表达量上调,CART表达量下调,以24 h最为显著,细胞凋亡率则随着复氧时间的延长而升高;应用此模型进行cAMP/PKA/CREB和NRSF-NRSE调控系统相互作用关系的研究中,我们在OGD处理SK-N-SH细胞3 h后,进行报告载体P-Luc-I、对照质粒pcDNA3.1或NRSF表达质粒pcDNA3.1-NRSF的共转染,荧光素酶活性检测的结果提示OGD处理可以引起NRSF表达的上调,但不足以抑制Forskolin/cAMP/PKA/CREB对CART的正性调控,当外源性转染NRSF表达质粒200ng、400ng/2×105个细胞时,则可以抑制Forskolin正性作用的发挥;细胞凋亡率的流式检测结果同样提示高水平的NRSF表达引起的NRSF-NRSE负性调控作用,可以抑制cAMP/PKA/CREB正性调控作用的发挥。 综上所述,我们通过生物信息学分析、电泳迁移率变动分析实验、染色质免疫共沉淀实验、荧光素酶报告系统活性检测证实了CART基因第一个内含子中的NRSE样基序通过与NRSF蛋白发生序列特异性结合从而发挥对CART基因的转录负调控作用,CART基因启动子、第一个内含子依赖于其各自区域内的NRSE元件之间的相互作用协同的发挥对CART基因的转录阻遏功能;高水平的NRSF表达引起的NRSF-NRSE负性调控作用可以有效的抑制cAMP/PKA/CREB正性调控系统作用的发挥。这一研究工作使我们对NRSF的转录调控作用、其对靶基因CART的转录调控机制研究又有了新的认识,为今后探讨该调控机制在干细胞向成熟神经元、胰岛细胞的发育分化及在病理损伤状态下,如何通过干预NRSF、CART基因的表达发挥治疗作用等方面提供了新的思路。
[Abstract]:Stem cells are a kind of cells with strong self-renewal and multi-directional differentiation potential. They can be induced to differentiate into mature neurons and islet cells under specific conditions in vitro, thus providing excellent seed cells for cell transplantation in the treatment of diabetes, Parkinson's syndrome, stroke and other diseases. However, they have important clinical application value. In the process of stem cells differentiating into neurons and islet cells, there are a series of problems, including inefficient differentiation, immature function of differentiated cells, etc. In the final analysis, the mechanism of differentiation is still unclear. NCER factor, NRSF; also known as RE-1 silencing transcription factor (REST) plays an important role in the differentiation of stem cells into neurons and islet cells. In addition, our laboratory studies have shown that NRSF regulates neuronal and islet-related target genes such as insulin, cocaine and amphetamine-regu. Based on this work, further study on the transcriptional regulation mechanism of NRSF on its target genes will help us better understand the role of NRSF in the differentiation of stem cells into neurons or islet cells and the maintenance of function of mature cells, so as to guide stem cells to mature nerves in the future. Cell or islet cell differentiation provides new ideas.
NRSF is a protein with nine Cys2/His2 zinc finger structures. It has one DNA binding domain, one lysine-rich domain, one proline-rich domain and two transcription inhibitory domains, N-terminal inhibitory domain and C-terminal inhibitory domain. It is widely expressed in embryonic stem cells (ES cells), neural stem cells (neural stem cells). Stem cells, NSCs, and non-neural cells, which are not expressed in most differentiated neurons, recruit different inhibitory complexes by binding to specific acting elements NRSE (neuron-restrictive silencer element, NRSE; also known as Repressor Element, RE1) to play a role in target genes such as type II sodium channel, SCG10, connectin 36 (Connex). In36) plays an important role in the maintenance of ES cell totipotency and self-renewal, early embryonic development, stem cell differentiation into neurons and islet cells.
CART is a new type of neuropeptide, which is distributed in the central and peripheral nervous system, adrenal glands, islets and other endocrine tissues. Many studies have shown that CART is involved in many physiological processes, such as food intake and obesity, stress, energy metabolism, neuroprotection and so on. The research mainly focuses on the localization, processing and function exertion of CART peptides. There are few studies on the transcriptional regulation of CART gene including transcription factors, regulatory elements and the neuroprotective mechanism of CART peptides. This system will provide powerful theoretical guidance for basic research and clinical treatment of diabetes, nervous system diseases and so on.
Our previous work has shown that CART gene is one of the target genes for NRSF. The NRSE-like motifs in the core promoter region of CART gene can specifically bind to NRSF sequence to regulate the transcriptional inhibition of CART gene. NRSE has a common NRSE-like motif that is very similar to each other and is conserved among species. Does this sequence also participate in the transcriptional regulation of CART gene? How does it coordinate with NRSE in the promoter region of CART gene to regulate the transcriptional regulation of CART gene? Only the positive regulatory pathway of cAMP/PKA/CREB is clear. So, how does the negative regulatory system of NRSF-NRSE and the positive regulatory system of cAMP/PKA/CREB compete to regulate the CART gene and jointly regulate the transcriptional level of CART gene?
This study mainly consists of three parts:
First, the transcriptional regulation of CART gene by intron sequence.
By using rVista software, we analyzed the intron sequences of human, rat and mouse CART genes retrieved from NCBI database of the National Center for Biomedical Information of the United States, and predicted a series of elements that might regulate the expression of CART genes, including ZF5, E47, NRSE, AREB6, and so on. Combining with previous work, we identified human, large and large. In order to investigate the transcriptional regulation of CART gene by the first intron of CART gene and its NRSE elements, we studied the NRSE-like motif of the first intron of CART gene in mice. Basic-CART32 (P-Luc), a luciferase reporter vector pGL3-Basic-CART32-Intron (P-Luc-I), pGL3-Basic-CART32-NRSEnon Intron (P-Luc-tI), was constructed and co-transfected into NTera2/CloneD1 cells with the luciferase-containing plasmid pRL-CMV. The results showed that the transcriptional activity of P-Luc-I was down-regulated by 57.9% (P 0.05) compared with the control group, indicating that the first intron sequence of CART gene was involved in the negative regulation of CART gene transcription. The activity of CART gene was up-regulated by 53.8% (P 0.05), suggesting that the intron of CART gene plays a negative role in the transcriptional regulation of CART gene through NRSE in its region. It was confirmed that the binding between NRSF and intron NRSE existed in the natural chromatin region.
Two, the regulatory effect of NRSF on CART gene transcription and its mechanism.
In the first part of this paper, we carried out a preliminary study on the NRSE-like sequence of the first intron region of the CART gene. In order to further explore how NRSF regulates the transcription of the gene through the promoter and intron sequences of the CART gene, we carried out a preliminary study on the NRSE-like sequence of the first intron region of the CART gene. Series of work. Electrophoretic mobility shift assay (EMSA) confirmed that the NRSE motif of CART gene promoter region and the first intron region could specifically bind to NRSF in vitro. Chromatin immunoprecipitation assay showed that NRSF and NRSE junction occurred in different NRSF expression cell lines. In order to understand how NRSF regulates the transcription of CART gene through promoter and intron sequences, we used CART promoter-luciferase reporter vectors P-Luc, P-Luc-tI, P-Lu. At the same time, we constructed the CART promoter-luciferase reporter vector pGL3-Basic-NRSEnon promoter (tP-Luc) and the luciferase reporter vector pGL3-Basic-NRSEnon promoter-N (tP-Luc-I), and the luciferase reporter vector pGL3-Basic-NRSEnon promoter-N (pGL3-Basic-NRSEnon Promoter-N) with or without NRSE motif deletion at the 3'end of the luciferase gene. RSEnon Intron (tP-Luc-tI), these reporter vectors were transfected into HeLa cells. The results of luciferase activity assay showed that CART gene intron and promoter cooperated with the negative transcriptional regulation of CART gene, and NRSF-NRSE in promoter region played a stronger negative transcriptional regulation on CART gene. Co-transfection Luciferase Report Similar results were also obtained in SK-N-SH cells with low NRSF expression plasmid pcDNA3.1-NRSF. In order to verify whether the negative transcriptional regulation of the CART gene promoter and the first intron depended on the NRSE elements in their respective regions, we originally contained the NRSE motif of the double-copy CART promoter (normal or normal). On the basis of the mutant sequence, a double-copy CART intron NRSE motif (normal or mutant) was inserted at the 3'-terminal of the luciferase gene. These luciferase reporter vectors were co-transfected into HeLa cells and SK-N-SH cells with the pRL-CMV plasmid containing marine luciferase, respectively. The results showed that the negative regulation of CART gene transcription by the promoter and the first intron region depended on the synergistic effect of NRSE elements in their respective regions.
Three, the positive and negative regulation system of CART gene expression and its mechanism of action.
To further investigate how the NRSF-NRSE negative regulatory system and the cAMP/PKA/CREB positive regulatory system competently regulate the expression of CART gene, we first co-transfected P-Luc-I and compared the plasmid pcDNA3.1-NRSF (100ng, 200ng, 400ng/2 *105 cells) with the plasmid pcDNA3.1 or the concentration gradient NRSF expression plasmid pcDNA3.1-NRSF (100ng, 200ng, 400ng/2 *105 cells) to detect the luciferase activity. The results showed that transfected pcDNA3.1-NRSF 400ng/2 *105 cells could inhibit the positive regulation of Forskolin/cAMP/PKA/CREB on CART, suggesting that the negative regulation of NRSF-NRSE induced by high level of NRSF expression could inhibit the function of positive regulation system of cAMP/PKA/CREB.
On this basis, we established an oxygen-glucose deprivation (OGD) model of neurons and carried out related experiments in combination with the function of CART peptides to reduce cell death induced by ischemia/hypoxia. In this model, we can further investigate whether changes in NRSF expression can cause changes in CART gene expression and affect the role of cAMP/PKA/CREB positive regulatory pathway.
SK-N-SH cells inoculated in 12-well plates were placed in a hypoxic glove box at 37 C, 0.3% O2, 95% N 2 for 3 hours to simulate the ischemia/hypoxia environment in vitro. The expression of NRSF and CART in the treated SK-N-SH cells was detected by RT-PCR and Western Blot after 24 h of reoxygenation, 48 h of reoxygenation, and the apoptosis rate was detected by flow cytometry. The results showed that the expression of NRSF was up-regulated and the expression of CART was down-regulated after reoxygenation in SK-N-SH cells treated with OGD, and the apoptosis rate was up-regulated with the prolongation of reoxygenation time. In the study of the interaction between cAMP/PKA/CREB and NRSF-NRSE regulatory system, we treated SK-N-SH cells with OGD for 3 hours. Comparing with the co-transfection of pcDNA3.1 or NRSF expression plasmid pcDNA3.1-NRSF, luciferase activity assay showed that OGD treatment could induce the up-regulation of NRSF expression, but could not inhibit the positive regulation of Forskolin/cAMP/PKA/CREB on CART. When exogenous transfection of NRSF expression plasmid 200 ng, 400 ng/2*105 cells The results of flow cytometry also indicated that the negative regulation of NRSF-NRSE induced by high level of NRSF expression could inhibit the positive regulation of cAMP/PKA/CREB.
In summary, we demonstrated that the NRSE-like motifs in the first intron of CART gene play a negative role in the transcriptional regulation of CART gene by specific binding to NRSF protein through bioinformatics analysis, electrophoretic mobility change assay, chromatin immunoprecipitation assay, and luciferase reporter system activity assay. CART gene promoter, the first intron, depends on the interaction of NRSE elements in their respective regions to play a synergistic role in the transcriptional repression of CART gene.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
本文编号：2219090
[Abstract]:Stem cells are a kind of cells with strong self-renewal and multi-directional differentiation potential. They can be induced to differentiate into mature neurons and islet cells under specific conditions in vitro, thus providing excellent seed cells for cell transplantation in the treatment of diabetes, Parkinson's syndrome, stroke and other diseases. However, they have important clinical application value. In the process of stem cells differentiating into neurons and islet cells, there are a series of problems, including inefficient differentiation, immature function of differentiated cells, etc. In the final analysis, the mechanism of differentiation is still unclear. NCER factor, NRSF; also known as RE-1 silencing transcription factor (REST) plays an important role in the differentiation of stem cells into neurons and islet cells. In addition, our laboratory studies have shown that NRSF regulates neuronal and islet-related target genes such as insulin, cocaine and amphetamine-regu. Based on this work, further study on the transcriptional regulation mechanism of NRSF on its target genes will help us better understand the role of NRSF in the differentiation of stem cells into neurons or islet cells and the maintenance of function of mature cells, so as to guide stem cells to mature nerves in the future. Cell or islet cell differentiation provides new ideas.
NRSF is a protein with nine Cys2/His2 zinc finger structures. It has one DNA binding domain, one lysine-rich domain, one proline-rich domain and two transcription inhibitory domains, N-terminal inhibitory domain and C-terminal inhibitory domain. It is widely expressed in embryonic stem cells (ES cells), neural stem cells (neural stem cells). Stem cells, NSCs, and non-neural cells, which are not expressed in most differentiated neurons, recruit different inhibitory complexes by binding to specific acting elements NRSE (neuron-restrictive silencer element, NRSE; also known as Repressor Element, RE1) to play a role in target genes such as type II sodium channel, SCG10, connectin 36 (Connex). In36) plays an important role in the maintenance of ES cell totipotency and self-renewal, early embryonic development, stem cell differentiation into neurons and islet cells.
CART is a new type of neuropeptide, which is distributed in the central and peripheral nervous system, adrenal glands, islets and other endocrine tissues. Many studies have shown that CART is involved in many physiological processes, such as food intake and obesity, stress, energy metabolism, neuroprotection and so on. The research mainly focuses on the localization, processing and function exertion of CART peptides. There are few studies on the transcriptional regulation of CART gene including transcription factors, regulatory elements and the neuroprotective mechanism of CART peptides. This system will provide powerful theoretical guidance for basic research and clinical treatment of diabetes, nervous system diseases and so on.
Our previous work has shown that CART gene is one of the target genes for NRSF. The NRSE-like motifs in the core promoter region of CART gene can specifically bind to NRSF sequence to regulate the transcriptional inhibition of CART gene. NRSE has a common NRSE-like motif that is very similar to each other and is conserved among species. Does this sequence also participate in the transcriptional regulation of CART gene? How does it coordinate with NRSE in the promoter region of CART gene to regulate the transcriptional regulation of CART gene? Only the positive regulatory pathway of cAMP/PKA/CREB is clear. So, how does the negative regulatory system of NRSF-NRSE and the positive regulatory system of cAMP/PKA/CREB compete to regulate the CART gene and jointly regulate the transcriptional level of CART gene?
This study mainly consists of three parts:
First, the transcriptional regulation of CART gene by intron sequence.
By using rVista software, we analyzed the intron sequences of human, rat and mouse CART genes retrieved from NCBI database of the National Center for Biomedical Information of the United States, and predicted a series of elements that might regulate the expression of CART genes, including ZF5, E47, NRSE, AREB6, and so on. Combining with previous work, we identified human, large and large. In order to investigate the transcriptional regulation of CART gene by the first intron of CART gene and its NRSE elements, we studied the NRSE-like motif of the first intron of CART gene in mice. Basic-CART32 (P-Luc), a luciferase reporter vector pGL3-Basic-CART32-Intron (P-Luc-I), pGL3-Basic-CART32-NRSEnon Intron (P-Luc-tI), was constructed and co-transfected into NTera2/CloneD1 cells with the luciferase-containing plasmid pRL-CMV. The results showed that the transcriptional activity of P-Luc-I was down-regulated by 57.9% (P 0.05) compared with the control group, indicating that the first intron sequence of CART gene was involved in the negative regulation of CART gene transcription. The activity of CART gene was up-regulated by 53.8% (P 0.05), suggesting that the intron of CART gene plays a negative role in the transcriptional regulation of CART gene through NRSE in its region. It was confirmed that the binding between NRSF and intron NRSE existed in the natural chromatin region.
Two, the regulatory effect of NRSF on CART gene transcription and its mechanism.
In the first part of this paper, we carried out a preliminary study on the NRSE-like sequence of the first intron region of the CART gene. In order to further explore how NRSF regulates the transcription of the gene through the promoter and intron sequences of the CART gene, we carried out a preliminary study on the NRSE-like sequence of the first intron region of the CART gene. Series of work. Electrophoretic mobility shift assay (EMSA) confirmed that the NRSE motif of CART gene promoter region and the first intron region could specifically bind to NRSF in vitro. Chromatin immunoprecipitation assay showed that NRSF and NRSE junction occurred in different NRSF expression cell lines. In order to understand how NRSF regulates the transcription of CART gene through promoter and intron sequences, we used CART promoter-luciferase reporter vectors P-Luc, P-Luc-tI, P-Lu. At the same time, we constructed the CART promoter-luciferase reporter vector pGL3-Basic-NRSEnon promoter (tP-Luc) and the luciferase reporter vector pGL3-Basic-NRSEnon promoter-N (tP-Luc-I), and the luciferase reporter vector pGL3-Basic-NRSEnon promoter-N (pGL3-Basic-NRSEnon Promoter-N) with or without NRSE motif deletion at the 3'end of the luciferase gene. RSEnon Intron (tP-Luc-tI), these reporter vectors were transfected into HeLa cells. The results of luciferase activity assay showed that CART gene intron and promoter cooperated with the negative transcriptional regulation of CART gene, and NRSF-NRSE in promoter region played a stronger negative transcriptional regulation on CART gene. Co-transfection Luciferase Report Similar results were also obtained in SK-N-SH cells with low NRSF expression plasmid pcDNA3.1-NRSF. In order to verify whether the negative transcriptional regulation of the CART gene promoter and the first intron depended on the NRSE elements in their respective regions, we originally contained the NRSE motif of the double-copy CART promoter (normal or normal). On the basis of the mutant sequence, a double-copy CART intron NRSE motif (normal or mutant) was inserted at the 3'-terminal of the luciferase gene. These luciferase reporter vectors were co-transfected into HeLa cells and SK-N-SH cells with the pRL-CMV plasmid containing marine luciferase, respectively. The results showed that the negative regulation of CART gene transcription by the promoter and the first intron region depended on the synergistic effect of NRSE elements in their respective regions.
Three, the positive and negative regulation system of CART gene expression and its mechanism of action.
To further investigate how the NRSF-NRSE negative regulatory system and the cAMP/PKA/CREB positive regulatory system competently regulate the expression of CART gene, we first co-transfected P-Luc-I and compared the plasmid pcDNA3.1-NRSF (100ng, 200ng, 400ng/2 *105 cells) with the plasmid pcDNA3.1 or the concentration gradient NRSF expression plasmid pcDNA3.1-NRSF (100ng, 200ng, 400ng/2 *105 cells) to detect the luciferase activity. The results showed that transfected pcDNA3.1-NRSF 400ng/2 *105 cells could inhibit the positive regulation of Forskolin/cAMP/PKA/CREB on CART, suggesting that the negative regulation of NRSF-NRSE induced by high level of NRSF expression could inhibit the function of positive regulation system of cAMP/PKA/CREB.
On this basis, we established an oxygen-glucose deprivation (OGD) model of neurons and carried out related experiments in combination with the function of CART peptides to reduce cell death induced by ischemia/hypoxia. In this model, we can further investigate whether changes in NRSF expression can cause changes in CART gene expression and affect the role of cAMP/PKA/CREB positive regulatory pathway.
SK-N-SH cells inoculated in 12-well plates were placed in a hypoxic glove box at 37 C, 0.3% O2, 95% N 2 for 3 hours to simulate the ischemia/hypoxia environment in vitro. The expression of NRSF and CART in the treated SK-N-SH cells was detected by RT-PCR and Western Blot after 24 h of reoxygenation, 48 h of reoxygenation, and the apoptosis rate was detected by flow cytometry. The results showed that the expression of NRSF was up-regulated and the expression of CART was down-regulated after reoxygenation in SK-N-SH cells treated with OGD, and the apoptosis rate was up-regulated with the prolongation of reoxygenation time. In the study of the interaction between cAMP/PKA/CREB and NRSF-NRSE regulatory system, we treated SK-N-SH cells with OGD for 3 hours. Comparing with the co-transfection of pcDNA3.1 or NRSF expression plasmid pcDNA3.1-NRSF, luciferase activity assay showed that OGD treatment could induce the up-regulation of NRSF expression, but could not inhibit the positive regulation of Forskolin/cAMP/PKA/CREB on CART. When exogenous transfection of NRSF expression plasmid 200 ng, 400 ng/2*105 cells The results of flow cytometry also indicated that the negative regulation of NRSF-NRSE induced by high level of NRSF expression could inhibit the positive regulation of cAMP/PKA/CREB.
In summary, we demonstrated that the NRSE-like motifs in the first intron of CART gene play a negative role in the transcriptional regulation of CART gene by specific binding to NRSF protein through bioinformatics analysis, electrophoretic mobility change assay, chromatin immunoprecipitation assay, and luciferase reporter system activity assay. CART gene promoter, the first intron, depends on the interaction of NRSE elements in their respective regions to play a synergistic role in the transcriptional repression of CART gene.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
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