EWSR1在有丝分裂中的作用机制研究
发布时间：2018-08-23 11:47
【摘要】：尤因肉瘤断点区域1蛋白(Ewing sarcoma breakpoint region 1 protein,EWSR1)是TET家族成员,属于RNA结合蛋白。TET家族蛋白除了可以直接结合RNA外,TET家族蛋白还可以结合DNA,也可以作为转录辅助因子直接与转录因子结合,参与包括基因表达调控、mRNA剪切编辑、信号通路调节、DNA修复等生物学过程。研究人员最早发现EWSR1是因为在尤因肉瘤和原始神经外胚层肿瘤相关亚型中发现22号和11号染色体的断点区产生了一个杂交转录本(t(ll;22)(q24;q12))。尤因肉瘤是发生在儿童和年轻成人中的第二常见的骨肉瘤,尤因肉瘤可以发生在任何年龄,但发病高峰期是在30岁以前,约5%的成人和10-15%的儿童发生此类肉瘤,并且没有性别差异。尤因肉瘤的治疗包括化疗、放疗和手术治疗,但不幸的是,肿瘤的预后很差,大量病例伴随着原发性肿瘤和肿瘤转移。超过90%的尤因肉瘤发生EWSR1与ETS家族基因FLI1和ERG的易位。这些易位形成的融合蛋白可以靶向细胞生长、增殖、衰老、以及肿瘤发生相关的基因,从而改变细胞的命运,导致增殖异常,产生不同类型的肿瘤。因此,关于EWSR1功能的研究主要集中在其与多种转录因子易位形成融合蛋白从而参与恶性肿瘤的发生发展的过程。EWSR1定位于染色体22q12.2上,编码含656个氨基酸,包括N端的转录激活结构域和C端的RNA结合结构域,C末端最后18个氨基酸是其核定位序列。EWSR1有6个不同的转录剪切本,经实验证实的仅有2个。EWSR1在进化上高度保守的,广泛表达于大多数组织和细胞中。EWSR1主要定位于细胞核,但是也可以定位在胞浆和细胞表面。后来有研究表明EWSR1在不同的细胞类型和不同细胞时期存在动态的亚细胞定位,在细胞分裂的不同时期出现动态分布。在细胞核内,EWSR1通过与转录因子TFIID、RNA聚合酶II(RNAP II)、转录激活子或抑制子相互作用参与转录调节过程,EWSR1可以与RNA和DNA结合发挥功能。EWSR1可以直接与剪切子结合影响剪切。EWSR1还通过结合非编码RNA调节基因表达。除了核内的功能,近年来开始有研究人员关注EWSR1的其他功能。EWSR1可能抑制微管解聚,影响细胞周期进程。EWSR1缺失导致有丝分裂缺陷和纺锤体异常,细胞凋亡增加。进一步研究表明EWS/FLi1结合并抑制EWSR1功能,EWSR1影响Aurora B在分裂后期中央区的定位从而使胞质分裂失败导致非整倍体产生。然而,EWSR1参与有丝分裂进程的分子机制还不完全清楚。本研究从ewsr1特异性的时空表达模式出发,检测了ewsr1的动态定位对细胞周期的影响,并围绕着ewsr1对纺锤体微管乙酰化的动态调节开展了分子机制的研究。我们的研究表明ewsr1在m期高表达,随着m期比例的增高,ewsr1呈全细胞分布的比例也增高,提示ewsr1在m期发挥重要作用。用nocodazole处理使细胞同步化到m期后释放,流式细胞术检测表明干涉ewsr1抑制细胞离开g2/m期,间接免疫荧光实验表明,干涉ewsr1延迟细胞离开m期。我们进一步统计了m期前中期和后末期细胞的比例,发现干涉ewsr1主要延迟细胞离开m期前中期的时间。接着我们通过time-lapse实验观察发现干涉ewsr1使细胞有丝分裂时间延长,并且主要使分裂前中期时间延长,而不影响后期时长。回转ewsr1-sirna不敏感的gfp-ewsr1可以逆转细胞延迟离开g2/m期的表型,并且逆转干涉ewsr1对细胞分裂前中期的延迟作用。为了更好地了解ewsr1调控细胞有丝分裂的机制,我们采用间接免疫荧光实验确定ewsr1在细胞周期各个时相的定位。在间期,ewsr1主要定位在细胞核;当细胞核膜破裂,细胞进入分裂前期时,ewsr1呈现全细胞分布;在细胞分裂中期、后期和末期,ewsr1定位在纺锤体上。用含taxol的pem处理明确了ewsr1与纺锤体的共定位。外源gfp-ewsr1也定位在纺锤体上,干涉ewsr1后ewsr1在纺锤体上的定位消失。随后,我们用免疫共沉淀实验检测到当细胞同步化到m期时ewsr1与纺锤体微管组分α-tubulin存在相互作用,而在非同步化的细胞中不存在相互作用。gstpull-down实验表明ewsr1与α-tubulin存在直接相互作用。回转ewsr1核定位序列缺失的ewsr1-sirna不敏感的myc-ewsr1Δnls质粒能够逆转干涉ewsr1对有丝分裂的延迟作用,用rna聚合酶ii的抑制剂α-amanitin抑制细胞转录活性不影响干涉ewsr1对有丝分裂进程的影响。提示ewsr1在有丝分裂中的作用不依赖于其核内的功能。微管再生实验表明干涉ewsr1抑制纺锤体微管的形成,而冷处理实验表明干涉ewsr1使纺锤体微管更不稳定,增加了微管对冷处理的敏感。bubr1和mad2是有丝分裂检查点sac蛋白复合体的重要组分,当微管与着丝粒结合拉着染色体排列到赤道板上,并达到一定张力时,bubr1和mad2信号失活,apc/c激活,染色体分离。我们的结果表明干涉ewsr1不影响bubr1和mad2在着丝粒上的定位。并且干涉ewsr1不改变aurorab分裂前期和中期的定位,表明aurorab不参与ewsr1对有丝分裂前中期的调控。微管组分α-Tubulin K40位的乙酰化与微管稳定性有密切关系。Western Blot实验表明在细胞同步化到M期时,过表达EWSR1促进α-Tubulin的乙酰化,干涉EWSR1抑制α-Tubulin的乙酰化,但是当细胞没有做同步化处理的时候,干涉EWSR1并不影响α-Tubulin的乙酰化。间接免疫荧光实验表明,干涉EWSR1抑制细胞分裂前期和中期α-Tubulin的乙酰化,而不影响其他时期,并且排除了染色体排列异常和MG132自身对乙酰化的影响。过表达核定位序列缺失的Myc-EWSR1ΔNLS仍能促进α-Tubulin的乙酰化,而α-amanitin处理细胞后干涉EWSR1仍能够抑制α-Tubulin的乙酰化,表明EWSR1的转录活性不影响α-Tubulin的乙酰化。干涉EWSR1不影响α-Tubulin的去酪氨酸化和多聚谷氨酸化修饰。HDAC6和SIRT2是已知的α-Tubulin去乙酰化酶。用HDAC6特异性抑制剂Tubacin抑制HDAC6活性或siRNA干涉HDAC6后再干涉EWSR1对α-Tubulin的乙酰化的抑制作用减弱。干涉SIRT2再干涉EWSR1仍能够抑制α-Tubulin的乙酰化,表明EWSR1通过HDAC6影响α-Tubulin的乙酰化而非SIRT2。Tubacin抑制HDAC6活性可以逆转干涉EWSR1对有丝分裂染色体排列异常和多极纺锤体的影响。免疫共沉淀表明EWSR1可能通过抑制HDAC6在纺锤体微管上的结合从而促进α-Tubulin的乙酰化。综上所述,我们的研究提示EWSR1在有丝分裂进程和纺锤体动态性调控中发挥重要作用,EWSR1通过HDAC6影响微管乙酰化,改变纺锤体动态性,从而影响有丝分裂进程。我们的研究进一步明确了EWSR1在有丝分裂进程中的作用,为靶向有丝分裂的肿瘤药物研发提供新的思路。
[Abstract]:Ewing sarcoma breakpoint region 1 protein (EWSR1) is a member of the TET family and belongs to the RNA-binding protein family. In addition to directly binding to RNA, TET family proteins can bind to DNA as well as directly binding to transcription factors, including gene expression regulation and mRNA. Researchers first discovered EWSR1 as a result of the discovery of a hybrid transcript (t (ll; 22) (q24; q12) at the breakpoint regions of chromosomes 22 and 11 in Ewing's sarcoma and primitive neuroectodermal tumor-related subtypes. The second most common type of osteosarcoma, Ewing's sarcoma, can occur at any age, but the peak is before 30 years of age. About 5% of adults and 10-15% of children develop this type of sarcoma without gender differences. Ewing's sarcoma treatment includes chemotherapy, radiotherapy and surgical treatment, but unfortunately, the prognosis of the tumor is poor, with a large number of cases accompanied by primary treatment. Sexual tumors and metastases. Over 90% of Ewing's sarcomas have translocations of EWSR1 and the ETS family genes FLI1 and ERG. These translocations form fusion proteins that target cell growth, proliferation, senescence, and genes associated with tumorigenesis, thereby altering cell fate, leading to abnormal proliferation and producing different types of tumors. EWSR1 is located on chromosome 22q12.2 and encodes 656 amino acids, including the N-terminal transcriptional activation domain and the C-terminal RNA binding domain. The last 18 amino acids at the C-terminal are the nucleotide localization sequences. WSR1 has six different transcription splices, only two of which have been proved highly conserved in evolution and widely expressed in most tissues and cells. EWSR1 is mainly located in the nucleus, but it can also be located in the cytoplasm and cell surface. Later studies have shown that EWSR1 is active in different cell types and different cell stages. In the nucleus, EWSR1 participates in transcriptional regulation by interacting with transcription factor TFIID, RNA polymerase II (RNAP II), transcription activator or inhibitor. EWSR1 can bind to RNA and DNA to perform its function. EWSR1 can bind directly to shears to influence shearing. SR1 also regulates gene expression by binding to non-coding RNA. In addition to nuclear functions, researchers have recently begun to focus on other functions of EWSR1. EWSR1 may inhibit microtubule depolymerization and affect cell cycle progression. EWSR1 affects the location of Aurora B in the central region of the anaphase leading to aneuploid production. However, the molecular mechanism of EWSR1's involvement in mitosis is still unclear. Our study shows that ewsr1 is highly expressed in M phase, and the proportion of ewsr1 in whole cell distribution increases with the increase of M phase ratio, suggesting that ewsr1 plays an important role in M phase. Cytological examination showed that interfering with ewsr1 inhibited the cell leaving g2/m phase. Indirect immunofluorescence assay showed that interfering with ewsr1 delayed the cell leaving m phase. We further calculated the proportions of cells in the pre-and post-M phase, and found that interfering with ewsr1 delayed the cell leaving the pre-and post-M phase mainly. Interference with ewsr1 prolonged the mitotic time of cells and prolonged the pre-and mid-mitotic time without affecting the late-mitotic time. Rotating ewsr1-sirna-insensitive gfp-ewsr1 reversed the delayed departure of g2/m phase and reversed the delayed effect of interfering with ewsr1 on the pre-and mid-mitotic phase of cells. We used indirect immunofluorescence assay to determine the location of ewsr1 at various stages of the cell cycle. During the interphase, ewsr1 was mainly located in the nucleus; when the nuclear membrane ruptured and the cell entered the prophase of division, ewsr1 was distributed throughout the cell; during the metaphase, anaphase and anaphase of cell division, ewsr1 was localized on the spindle. Exogenous gfp-ewsr1 was also localized on the spindle. After interfering with ewsr1, the localization of ewsr1 disappeared on the spindle. Subsequently, we detected the interaction between ewsr1 and spindle microtubule component alpha-tubulin when the cells reached phase m by immunoprecipitation assay. Gstpull-down assay showed that there was a direct interaction between ewsr1 and alpha-tubulin. Myc-ewsr1 NLS plasmid, which was insensitive to rotated ewsr1-sirna, could reverse the delayed mitotic effect of interfering with ewsr1, and alpha-amanitin, an inhibitor of RNA polymerase ii, could inhibit the transcriptional activity of cells. It is suggested that the role of ewsr1 in mitosis does not depend on its nuclear function. Microtubule regeneration experiments show that the interference of ewsr1 inhibits the formation of spindle microtubules. Cold treatment experiments show that the interference of ewsr1 makes spindle microtubules more unstable and increases the sensitivity of microtubules to cold treatment. When the microtubules bind to the centromere and pull the chromosomes to align on the equatorial plate and reach a certain tension, BubR1 and Mad2 signals are inactivated, APC / C is activated, and chromosomes are segregated. Our results show that interference with ewsr1 does not affect the localization of BubR1 and Mad2 on the centromere. The localization of aurorab in the prophase and metaphase of mitosis indicates that aurorab is not involved in the regulation of ewsr1 on the prophase and metaphase of mitosis. Acetylation of the microtubule component at the position of alpha-Tubulin K40 is closely related to microtubule stability. Western Blot assay shows that over-expression of EWSR1 promotes the acetylation of alpha-Tubulin and interferes with the inhibition of alpha-Tubuli by EWSR1 at the time of cell synchronization to M phase. Interference with EWSR1 did not affect the acetylation of alpha-Tubulin when the cells were not synchronized. Indirect immunofluorescence assays showed that interfering with EWSR1 inhibited the acetylation of alpha-Tubulin in the prophase and metaphase of cell division without affecting other stages, and excluded chromosomal aberration and MG132's own acetylation. Overexpression of Myc-EWSR1_NLS could still promote the acetylation of alpha-Tubulin, while interfering with EWSR1 could inhibit the acetylation of alpha-Tubulin after treatment with alpha-amanitin, suggesting that the transcriptional activity of EWSR1 did not affect the acetylation of alpha-Tubulin. Interfering with EWSR1 did not affect the de-tyrosination and polyglutamic acid repair of alpha-Tubulin. HDAC6 and SIRT2 are known as alpha-Tubulin deacetylases. Inhibition of HDAC6 activity by Tubacin, a specific inhibitor of HDAC6, or inhibition of alpha-Tubulin acetylation by interfering with EWSR1 after interfering with HDAC6 by siRNA is weakened. Interference with SIRT2 and interfering with EWSR1 can still inhibit the acetylation of alpha-Tubulin, suggesting that EWSR1 affects the acetylation of alpha-Tubulin through HDAC6. Inhibition of HDAC6 activity by chemical rather than SIRT2.Tubacin reverses interference with EWSR1 in mitotic chromosomal aberration and multipolar spindles.Immunocoprecipitation suggests that EWSR1 may promote the acetylation of alpha-Tubulin by inhibiting HDAC6 binding to spindle microtubules. EWSR1 affects microtubule acetylation and spindle dynamics through HDAC6, thus affecting mitosis. Our study further clarifies the role of EWSR1 in mitosis and provides new ideas for the development of cancer drugs targeting mitosis.
【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】：2016
【分类号】：R73-3
本文编号：2198991
[Abstract]:Ewing sarcoma breakpoint region 1 protein (EWSR1) is a member of the TET family and belongs to the RNA-binding protein family. In addition to directly binding to RNA, TET family proteins can bind to DNA as well as directly binding to transcription factors, including gene expression regulation and mRNA. Researchers first discovered EWSR1 as a result of the discovery of a hybrid transcript (t (ll; 22) (q24; q12) at the breakpoint regions of chromosomes 22 and 11 in Ewing's sarcoma and primitive neuroectodermal tumor-related subtypes. The second most common type of osteosarcoma, Ewing's sarcoma, can occur at any age, but the peak is before 30 years of age. About 5% of adults and 10-15% of children develop this type of sarcoma without gender differences. Ewing's sarcoma treatment includes chemotherapy, radiotherapy and surgical treatment, but unfortunately, the prognosis of the tumor is poor, with a large number of cases accompanied by primary treatment. Sexual tumors and metastases. Over 90% of Ewing's sarcomas have translocations of EWSR1 and the ETS family genes FLI1 and ERG. These translocations form fusion proteins that target cell growth, proliferation, senescence, and genes associated with tumorigenesis, thereby altering cell fate, leading to abnormal proliferation and producing different types of tumors. EWSR1 is located on chromosome 22q12.2 and encodes 656 amino acids, including the N-terminal transcriptional activation domain and the C-terminal RNA binding domain. The last 18 amino acids at the C-terminal are the nucleotide localization sequences. WSR1 has six different transcription splices, only two of which have been proved highly conserved in evolution and widely expressed in most tissues and cells. EWSR1 is mainly located in the nucleus, but it can also be located in the cytoplasm and cell surface. Later studies have shown that EWSR1 is active in different cell types and different cell stages. In the nucleus, EWSR1 participates in transcriptional regulation by interacting with transcription factor TFIID, RNA polymerase II (RNAP II), transcription activator or inhibitor. EWSR1 can bind to RNA and DNA to perform its function. EWSR1 can bind directly to shears to influence shearing. SR1 also regulates gene expression by binding to non-coding RNA. In addition to nuclear functions, researchers have recently begun to focus on other functions of EWSR1. EWSR1 may inhibit microtubule depolymerization and affect cell cycle progression. EWSR1 affects the location of Aurora B in the central region of the anaphase leading to aneuploid production. However, the molecular mechanism of EWSR1's involvement in mitosis is still unclear. Our study shows that ewsr1 is highly expressed in M phase, and the proportion of ewsr1 in whole cell distribution increases with the increase of M phase ratio, suggesting that ewsr1 plays an important role in M phase. Cytological examination showed that interfering with ewsr1 inhibited the cell leaving g2/m phase. Indirect immunofluorescence assay showed that interfering with ewsr1 delayed the cell leaving m phase. We further calculated the proportions of cells in the pre-and post-M phase, and found that interfering with ewsr1 delayed the cell leaving the pre-and post-M phase mainly. Interference with ewsr1 prolonged the mitotic time of cells and prolonged the pre-and mid-mitotic time without affecting the late-mitotic time. Rotating ewsr1-sirna-insensitive gfp-ewsr1 reversed the delayed departure of g2/m phase and reversed the delayed effect of interfering with ewsr1 on the pre-and mid-mitotic phase of cells. We used indirect immunofluorescence assay to determine the location of ewsr1 at various stages of the cell cycle. During the interphase, ewsr1 was mainly located in the nucleus; when the nuclear membrane ruptured and the cell entered the prophase of division, ewsr1 was distributed throughout the cell; during the metaphase, anaphase and anaphase of cell division, ewsr1 was localized on the spindle. Exogenous gfp-ewsr1 was also localized on the spindle. After interfering with ewsr1, the localization of ewsr1 disappeared on the spindle. Subsequently, we detected the interaction between ewsr1 and spindle microtubule component alpha-tubulin when the cells reached phase m by immunoprecipitation assay. Gstpull-down assay showed that there was a direct interaction between ewsr1 and alpha-tubulin. Myc-ewsr1 NLS plasmid, which was insensitive to rotated ewsr1-sirna, could reverse the delayed mitotic effect of interfering with ewsr1, and alpha-amanitin, an inhibitor of RNA polymerase ii, could inhibit the transcriptional activity of cells. It is suggested that the role of ewsr1 in mitosis does not depend on its nuclear function. Microtubule regeneration experiments show that the interference of ewsr1 inhibits the formation of spindle microtubules. Cold treatment experiments show that the interference of ewsr1 makes spindle microtubules more unstable and increases the sensitivity of microtubules to cold treatment. When the microtubules bind to the centromere and pull the chromosomes to align on the equatorial plate and reach a certain tension, BubR1 and Mad2 signals are inactivated, APC / C is activated, and chromosomes are segregated. Our results show that interference with ewsr1 does not affect the localization of BubR1 and Mad2 on the centromere. The localization of aurorab in the prophase and metaphase of mitosis indicates that aurorab is not involved in the regulation of ewsr1 on the prophase and metaphase of mitosis. Acetylation of the microtubule component at the position of alpha-Tubulin K40 is closely related to microtubule stability. Western Blot assay shows that over-expression of EWSR1 promotes the acetylation of alpha-Tubulin and interferes with the inhibition of alpha-Tubuli by EWSR1 at the time of cell synchronization to M phase. Interference with EWSR1 did not affect the acetylation of alpha-Tubulin when the cells were not synchronized. Indirect immunofluorescence assays showed that interfering with EWSR1 inhibited the acetylation of alpha-Tubulin in the prophase and metaphase of cell division without affecting other stages, and excluded chromosomal aberration and MG132's own acetylation. Overexpression of Myc-EWSR1_NLS could still promote the acetylation of alpha-Tubulin, while interfering with EWSR1 could inhibit the acetylation of alpha-Tubulin after treatment with alpha-amanitin, suggesting that the transcriptional activity of EWSR1 did not affect the acetylation of alpha-Tubulin. Interfering with EWSR1 did not affect the de-tyrosination and polyglutamic acid repair of alpha-Tubulin. HDAC6 and SIRT2 are known as alpha-Tubulin deacetylases. Inhibition of HDAC6 activity by Tubacin, a specific inhibitor of HDAC6, or inhibition of alpha-Tubulin acetylation by interfering with EWSR1 after interfering with HDAC6 by siRNA is weakened. Interference with SIRT2 and interfering with EWSR1 can still inhibit the acetylation of alpha-Tubulin, suggesting that EWSR1 affects the acetylation of alpha-Tubulin through HDAC6. Inhibition of HDAC6 activity by chemical rather than SIRT2.Tubacin reverses interference with EWSR1 in mitotic chromosomal aberration and multipolar spindles.Immunocoprecipitation suggests that EWSR1 may promote the acetylation of alpha-Tubulin by inhibiting HDAC6 binding to spindle microtubules. EWSR1 affects microtubule acetylation and spindle dynamics through HDAC6, thus affecting mitosis. Our study further clarifies the role of EWSR1 in mitosis and provides new ideas for the development of cancer drugs targeting mitosis.
【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】：2016
【分类号】：R73-3
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1 王易龙;EWSR1在有丝分裂中的作用机制研究[D];中国人民解放军军事医学科学院;2016年
，本文编号：2198991
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