EWSR1在有絲分裂中的作用機制研究
發(fā)布時間：2018-08-23 11:47
【摘要】：尤因肉瘤斷點區(qū)域1蛋白(Ewing sarcoma breakpoint region 1 protein,EWSR1)是TET家族成員,屬于RNA結合蛋白。TET家族蛋白除了可以直接結合RNA外,TET家族蛋白還可以結合DNA,也可以作為轉錄輔助因子直接與轉錄因子結合,參與包括基因表達調(diào)控、mRNA剪切編輯、信號通路調(diào)節(jié)、DNA修復等生物學過程。研究人員最早發(fā)現(xiàn)EWSR1是因為在尤因肉瘤和原始神經(jīng)外胚層腫瘤相關亞型中發(fā)現(xiàn)22號和11號染色體的斷點區(qū)產(chǎn)生了一個雜交轉錄本(t(ll;22)(q24;q12))。尤因肉瘤是發(fā)生在兒童和年輕成人中的第二常見的骨肉瘤,尤因肉瘤可以發(fā)生在任何年齡,但發(fā)病高峰期是在30歲以前,約5%的成人和10-15%的兒童發(fā)生此類肉瘤,并且沒有性別差異。尤因肉瘤的治療包括化療、放療和手術治療,但不幸的是,腫瘤的預后很差,大量病例伴隨著原發(fā)性腫瘤和腫瘤轉移。超過90%的尤因肉瘤發(fā)生EWSR1與ETS家族基因FLI1和ERG的易位。這些易位形成的融合蛋白可以靶向細胞生長、增殖、衰老、以及腫瘤發(fā)生相關的基因,從而改變細胞的命運,導致增殖異常,產(chǎn)生不同類型的腫瘤。因此,關于EWSR1功能的研究主要集中在其與多種轉錄因子易位形成融合蛋白從而參與惡性腫瘤的發(fā)生發(fā)展的過程。EWSR1定位于染色體22q12.2上,編碼含656個氨基酸,包括N端的轉錄激活結構域和C端的RNA結合結構域,C末端最后18個氨基酸是其核定位序列。EWSR1有6個不同的轉錄剪切本,經(jīng)實驗證實的僅有2個。EWSR1在進化上高度保守的,廣泛表達于大多數(shù)組織和細胞中。EWSR1主要定位于細胞核,但是也可以定位在胞漿和細胞表面。后來有研究表明EWSR1在不同的細胞類型和不同細胞時期存在動態(tài)的亞細胞定位,在細胞分裂的不同時期出現(xiàn)動態(tài)分布。在細胞核內(nèi),EWSR1通過與轉錄因子TFIID、RNA聚合酶II(RNAP II)、轉錄激活子或抑制子相互作用參與轉錄調(diào)節(jié)過程,EWSR1可以與RNA和DNA結合發(fā)揮功能。EWSR1可以直接與剪切子結合影響剪切。EWSR1還通過結合非編碼RNA調(diào)節(jié)基因表達。除了核內(nèi)的功能,近年來開始有研究人員關注EWSR1的其他功能。EWSR1可能抑制微管解聚,影響細胞周期進程。EWSR1缺失導致有絲分裂缺陷和紡錘體異常,細胞凋亡增加。進一步研究表明EWS/FLi1結合并抑制EWSR1功能,EWSR1影響Aurora B在分裂后期中央?yún)^(qū)的定位從而使胞質(zhì)分裂失敗導致非整倍體產(chǎn)生。然而,EWSR1參與有絲分裂進程的分子機制還不完全清楚。本研究從ewsr1特異性的時空表達模式出發(fā),檢測了ewsr1的動態(tài)定位對細胞周期的影響,并圍繞著ewsr1對紡錘體微管乙酰化的動態(tài)調(diào)節(jié)開展了分子機制的研究。我們的研究表明ewsr1在m期高表達,隨著m期比例的增高,ewsr1呈全細胞分布的比例也增高,提示ewsr1在m期發(fā)揮重要作用。用nocodazole處理使細胞同步化到m期后釋放,流式細胞術檢測表明干涉ewsr1抑制細胞離開g2/m期,間接免疫熒光實驗表明,干涉ewsr1延遲細胞離開m期。我們進一步統(tǒng)計了m期前中期和后末期細胞的比例,發(fā)現(xiàn)干涉ewsr1主要延遲細胞離開m期前中期的時間。接著我們通過time-lapse實驗觀察發(fā)現(xiàn)干涉ewsr1使細胞有絲分裂時間延長,并且主要使分裂前中期時間延長,而不影響后期時長�；剞Dewsr1-sirna不敏感的gfp-ewsr1可以逆轉細胞延遲離開g2/m期的表型,并且逆轉干涉ewsr1對細胞分裂前中期的延遲作用。為了更好地了解ewsr1調(diào)控細胞有絲分裂的機制,我們采用間接免疫熒光實驗確定ewsr1在細胞周期各個時相的定位。在間期,ewsr1主要定位在細胞核;當細胞核膜破裂,細胞進入分裂前期時,ewsr1呈現(xiàn)全細胞分布;在細胞分裂中期、后期和末期,ewsr1定位在紡錘體上。用含taxol的pem處理明確了ewsr1與紡錘體的共定位。外源gfp-ewsr1也定位在紡錘體上,干涉ewsr1后ewsr1在紡錘體上的定位消失。隨后,我們用免疫共沉淀實驗檢測到當細胞同步化到m期時ewsr1與紡錘體微管組分α-tubulin存在相互作用,而在非同步化的細胞中不存在相互作用。gstpull-down實驗表明ewsr1與α-tubulin存在直接相互作用。回轉ewsr1核定位序列缺失的ewsr1-sirna不敏感的myc-ewsr1Δnls質(zhì)粒能夠逆轉干涉ewsr1對有絲分裂的延遲作用,用rna聚合酶ii的抑制劑α-amanitin抑制細胞轉錄活性不影響干涉ewsr1對有絲分裂進程的影響。提示ewsr1在有絲分裂中的作用不依賴于其核內(nèi)的功能。微管再生實驗表明干涉ewsr1抑制紡錘體微管的形成,而冷處理實驗表明干涉ewsr1使紡錘體微管更不穩(wěn)定,增加了微管對冷處理的敏感。bubr1和mad2是有絲分裂檢查點sac蛋白復合體的重要組分,當微管與著絲粒結合拉著染色體排列到赤道板上,并達到一定張力時,bubr1和mad2信號失活,apc/c激活,染色體分離。我們的結果表明干涉ewsr1不影響bubr1和mad2在著絲粒上的定位。并且干涉ewsr1不改變aurorab分裂前期和中期的定位,表明aurorab不參與ewsr1對有絲分裂前中期的調(diào)控。微管組分α-Tubulin K40位的乙酰化與微管穩(wěn)定性有密切關系。Western Blot實驗表明在細胞同步化到M期時,過表達EWSR1促進α-Tubulin的乙�；�,干涉EWSR1抑制α-Tubulin的乙�；�,但是當細胞沒有做同步化處理的時候,干涉EWSR1并不影響α-Tubulin的乙�；�。間接免疫熒光實驗表明,干涉EWSR1抑制細胞分裂前期和中期α-Tubulin的乙酰化,而不影響其他時期,并且排除了染色體排列異常和MG132自身對乙�；挠绊�。過表達核定位序列缺失的Myc-EWSR1ΔNLS仍能促進α-Tubulin的乙�；�,而α-amanitin處理細胞后干涉EWSR1仍能夠抑制α-Tubulin的乙�；�,表明EWSR1的轉錄活性不影響α-Tubulin的乙酰化。干涉EWSR1不影響α-Tubulin的去酪氨酸化和多聚谷氨酸化修飾。HDAC6和SIRT2是已知的α-Tubulin去乙酰化酶。用HDAC6特異性抑制劑Tubacin抑制HDAC6活性或siRNA干涉HDAC6后再干涉EWSR1對α-Tubulin的乙�；囊种谱饔脺p弱。干涉SIRT2再干涉EWSR1仍能夠抑制α-Tubulin的乙�；�,表明EWSR1通過HDAC6影響α-Tubulin的乙�；荢IRT2。Tubacin抑制HDAC6活性可以逆轉干涉EWSR1對有絲分裂染色體排列異常和多極紡錘體的影響。免疫共沉淀表明EWSR1可能通過抑制HDAC6在紡錘體微管上的結合從而促進α-Tubulin的乙�；�。綜上所述,我們的研究提示EWSR1在有絲分裂進程和紡錘體動態(tài)性調(diào)控中發(fā)揮重要作用,EWSR1通過HDAC6影響微管乙�；�,改變紡錘體動態(tài)性,從而影響有絲分裂進程。我們的研究進一步明確了EWSR1在有絲分裂進程中的作用,為靶向有絲分裂的腫瘤藥物研發(fā)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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R73-3
本文編號：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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R73-3
【相似文獻】
相關博士學位論文 前1條
1 王易龍;EWSR1在有絲分裂中的作用機制研究[D];中國人民解放軍軍事醫(yī)學科學院;2016年
，本文編號：2198991
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