ZNF496在乳腺癌發(fā)生發(fā)展中功能與作用機(jī)制的研究
發(fā)布時(shí)間：2018-09-10 07:14
【摘要】：雌激素受體α(estrogen receptor alpha,ERα)屬于核受體超家族的一員,參與多種生理學(xué)和病理學(xué)過(guò)程。雌激素結(jié)合活化的ERα可形成同源二聚體,與回文的雌激素反應(yīng)元件(estrogen response element,ERE)結(jié)合,發(fā)揮轉(zhuǎn)錄激活的功能。ERα主要包含3個(gè)重要的結(jié)構(gòu)域,分別是AF1結(jié)構(gòu)域、AF2結(jié)構(gòu)域和DBD結(jié)構(gòu)域。AF1和AF2都是轉(zhuǎn)錄激活結(jié)構(gòu)域,但只有AF2結(jié)構(gòu)域具有雌激素依賴(lài)性。DBD結(jié)構(gòu)域主要介導(dǎo)ERα與DNA序列的相互作用。乳腺癌作為女性發(fā)病率最高的惡性腫瘤之一,具有十分復(fù)雜的發(fā)病機(jī)制。目前認(rèn)為,人體內(nèi)分泌水平的異常是誘發(fā)乳腺癌的一個(gè)重要原因。約3/4的乳腺癌為ERα陽(yáng)性,而ERα的過(guò)度活化能夠刺激正常細(xì)胞的惡性轉(zhuǎn)化,也是維持乳腺癌細(xì)胞增殖的一個(gè)必要條件。ERα與配體和共調(diào)節(jié)因子結(jié)合的失調(diào)以及自身結(jié)構(gòu)和翻譯后化修飾的變化都會(huì)不同程度地影響雌激素受體的信號(hào)通路,參與乳腺癌的發(fā)生發(fā)展。因此,ERα是乳腺癌的生物標(biāo)記物,常用于指導(dǎo)乳腺癌的治療。目前,靶向ERα是乳腺癌輔助治療中最常用的策略之一。Tamoxifen是ERα的一個(gè)經(jīng)典拮抗劑,通過(guò)與雌激素競(jìng)爭(zhēng)結(jié)合ERα,抑制ERα的活性,抑制乳腺癌細(xì)胞的生長(zhǎng)和增殖。同時(shí),ERα共調(diào)節(jié)因子的豐度和/或活性的異常往往與乳腺癌的發(fā)生率和致死率具有相關(guān)性,也是預(yù)測(cè)和治療乳腺癌的潛在靶標(biāo),因此對(duì)ERα調(diào)節(jié)蛋白的研究對(duì)揭示ERα的調(diào)控機(jī)制,鑒定乳腺癌的診斷和治療靶標(biāo)具有重要的意義。C2H2型鋅指蛋白家族是哺乳動(dòng)物中最大的轉(zhuǎn)錄/轉(zhuǎn)錄調(diào)控因子家族,而KRAB型鋅指蛋白只存在于四足脊椎動(dòng)物中,具有相似的結(jié)構(gòu)和作用機(jī)制,且隨著物種進(jìn)化數(shù)量急劇增加,在人類(lèi)基因組中數(shù)量高達(dá)423種,占據(jù)了C2H2型鋅指蛋白總數(shù)的60%左右。KRAB型鋅指蛋白N端的KRAB結(jié)構(gòu)域通過(guò)與KAP1相互作用招募轉(zhuǎn)錄抑制蛋白,形成轉(zhuǎn)錄沉默復(fù)合物,發(fā)揮轉(zhuǎn)錄抑制的功能,而C端的C2H2型鋅指結(jié)構(gòu)域主要介導(dǎo)與特定DNA序列或其他轉(zhuǎn)錄因子的結(jié)合,將轉(zhuǎn)錄沉默復(fù)合物錨定在特定染色質(zhì)位點(diǎn)。越來(lái)越多的證據(jù)表明,KRAB型鋅指蛋白的出現(xiàn)和數(shù)量膨脹與脊椎動(dòng)物的進(jìn)化及其精細(xì)轉(zhuǎn)錄調(diào)控網(wǎng)絡(luò)的建立和完善密切相關(guān)。KRAB型鋅指蛋白參與穩(wěn)定基因組結(jié)構(gòu)、胚胎發(fā)育、細(xì)胞的增殖和分化等正常生理活動(dòng),又與腫瘤的發(fā)生發(fā)展有某些關(guān)聯(lián)。一般情況下,癌細(xì)胞中轉(zhuǎn)錄調(diào)控網(wǎng)絡(luò)趨于簡(jiǎn)單化,而癌組織中KRAB型鋅指蛋白的豐度也往往低于癌旁組織。ZNF496屬于KRAB型鋅指蛋白,可通過(guò)其C2HR結(jié)構(gòu)域與NSD1(nuclear receptor binding SET domain protein 1)結(jié)合,介導(dǎo)KAP-1不依賴(lài)的轉(zhuǎn)錄抑制功能。目前,尚未有關(guān)于ZNF496功能及機(jī)制的詳細(xì)報(bào)道。通過(guò)www.proteinatlas.org網(wǎng)站對(duì)其表達(dá)譜分析,發(fā)現(xiàn)ZNF496在女性生殖系統(tǒng)和乳腺中高表達(dá),但是在相應(yīng)的癌組織中表達(dá)很少或不表達(dá),提示ZNF496可能與乳腺癌的發(fā)生發(fā)展有關(guān)聯(lián),但是其中的機(jī)制尚不明確。我們的研究發(fā)現(xiàn):1、ZNF496在女性乳腺和生殖系統(tǒng)中高表達(dá),在相應(yīng)的癌組織中低表達(dá)。我們通過(guò)購(gòu)買(mǎi)的人類(lèi)生殖系統(tǒng)腫瘤組織芯片發(fā)現(xiàn),ZNF496在乳腺癌、卵巢癌和宮頸癌中的表達(dá)低于癌旁組織。我們還通過(guò)WB檢測(cè)了成年雄鼠和雌鼠的主要組織器官中ZNF496的相對(duì)表達(dá)豐度。結(jié)果顯示ZNF496在肺、卵巢、輸卵管和子宮中特異性表達(dá)。ZNF496的這種組織特異性表達(dá)可能與這些組織器官的正常生理功能的維持相關(guān)。2、ZNF496能夠與ERα相互作用:為了探討ZNF496的功能機(jī)制,我們首先通過(guò)IP-MS鑒定MCF-7細(xì)胞核中ZNF496的潛在相互作用蛋白,質(zhì)譜結(jié)果顯示ERα可能是ZNF496潛在的相互作用蛋白。隨后的Co-IP證實(shí),無(wú)論E2是否存在,ZNF496均能夠與ERα發(fā)生相互作用。3、ZNF496選擇性抑制ERα的轉(zhuǎn)錄活性:qPCR實(shí)驗(yàn)證實(shí),在E2處理?xiàng)l件下,ZNF496的過(guò)表達(dá)能夠抑制,敲低能夠促進(jìn)ERα靶基因Greb1、pS2、Xbp1、Sgk1和Wisp2的轉(zhuǎn)錄水平,而對(duì)ERα另一個(gè)靶基因Serpina3的表達(dá)沒(méi)有影響,同時(shí)ZNF496并不影響ERα的mRNA和蛋白水平。這說(shuō)明了ZNF496能夠選擇性調(diào)控ERα的轉(zhuǎn)錄活性。4、ZNF496選擇性抑制ERα與部分靶基因啟動(dòng)子的結(jié)合:實(shí)驗(yàn)室他人工作證實(shí)ZNF496蛋白C端的C2H2型鋅指結(jié)構(gòu)域和ERα蛋白中部的DBD結(jié)構(gòu)域介導(dǎo)了它們之間的相互作用,提示ZNF496可能影響ERα與ERE區(qū)的結(jié)合。熒光定位實(shí)驗(yàn)證實(shí),在E2處理?xiàng)l件下,ZNF496的過(guò)表達(dá)減弱活化的ERα在核內(nèi)的凝集現(xiàn)象。為了探討ZNF496選擇性調(diào)控ERα的機(jī)制,我們開(kāi)展了EMSA實(shí)驗(yàn)。結(jié)果顯示,在E2處理?xiàng)l件下,隨著ZNF496表達(dá)量的增加,ERα結(jié)合ERE序列的能力逐漸減弱。進(jìn)一步提示,在E2處理?xiàng)l件下,ZNF496的過(guò)表達(dá)能夠抑制ERα與ERE序列的結(jié)合。最后我們利用Ch IP-qPCR實(shí)驗(yàn)檢測(cè)MCF-7中內(nèi)源ERα與其靶基因啟動(dòng)子區(qū)域的結(jié)合能力。結(jié)果顯示,在E2處理?xiàng)l件下,ZNF496過(guò)表達(dá)之后,ERα結(jié)合pS2、Greb1和Wisp2等基因啟動(dòng)子的能力出現(xiàn)減弱趨勢(shì),而ERα結(jié)合Serpina3啟動(dòng)子的能力沒(méi)有變化。5、ZNF496可抑制ERα陽(yáng)性乳腺癌細(xì)胞的增殖:利用慢病毒感染,獲得ZNF496穩(wěn)定過(guò)表達(dá)的細(xì)胞株MCF-7、T-47D和MDA-MB-231。CCK-8測(cè)細(xì)胞增殖實(shí)驗(yàn)顯示,在E2處理?xiàng)l件下,ZNF496過(guò)表達(dá)能夠抑制MCF-7和T-47D的增殖,而對(duì)MDA-MB-231沒(méi)影響�？寺⌒纬蓪�(shí)驗(yàn)也證實(shí),在正常培養(yǎng)基培養(yǎng)條件下,ZNF496過(guò)表達(dá)能夠抑制MCF-7和T-47D的克隆形成能力,而對(duì)MDA-MB-231沒(méi)影響。因此,ZNF496通過(guò)選擇性抑制ERα的活性抑制ERα陽(yáng)性乳腺癌細(xì)胞的增殖。6、ZNF496在乳腺癌中的差異表達(dá)依賴(lài)于ERα的狀態(tài):29例乳腺癌病例樣本的免疫組化結(jié)果顯示,ZNF496在癌組織中的表達(dá)顯著低于癌旁組織。當(dāng)我們將乳腺癌樣本劃分為ERα+和ERα-時(shí),ZNF496在ERα+乳腺癌組織中的表達(dá)極顯著低于癌旁組織,而ZNF496在ERα-乳腺癌組織中的表達(dá)與癌旁組織沒(méi)有顯著性差異。因此ZNF496在乳腺癌中的差異表達(dá)依賴(lài)于ERα的陽(yáng)性表達(dá)。綜上所述,本研究發(fā)現(xiàn)ZNF496通過(guò)競(jìng)爭(zhēng)結(jié)合ERα的DBD結(jié)構(gòu)域選擇性抑制ERα與ERE區(qū)的結(jié)合,降低ERα的轉(zhuǎn)錄活性。ZNF496依賴(lài)對(duì)ERα的調(diào)控發(fā)揮抑制ERα陽(yáng)性乳腺癌細(xì)胞增殖的作用。本研究為進(jìn)一步揭示ERα陽(yáng)性乳腺癌發(fā)生發(fā)展的分子機(jī)理提供了思路,為乳腺癌的診斷或治療提供了潛在靶標(biāo),同時(shí)完善了人們對(duì)KRAB型鋅指蛋白家族成員功能與作用機(jī)制的認(rèn)識(shí)。
[Abstract]:Estrogen receptor alpha (ERa), a member of the nuclear receptor superfamily, is involved in a variety of physiological and pathological processes. ERa, which is activated by estrogen binding, can form homologous dimers and bind to palindromic estrogen response elements (EREs) to activate transcription. The AF1 domain, AF2 domain and DBD domain are all transcription-activated domains, but only AF2 domain is estrogen-dependent. Abnormal endocrine levels are thought to be an important cause of breast cancer. About 3/4 of breast cancers are ER-alpha-positive, and excessive activation of ER-alpha can stimulate malignant transformation of normal cells, which is also a necessary condition for maintaining the proliferation of breast cancer cells. The changes of posttranslational modification can affect the signal pathway of estrogen receptor and participate in the occurrence and development of breast cancer to varying degrees. Therefore, ERa is a biomarker of breast cancer and is often used to guide the treatment of breast cancer. It can inhibit the growth and proliferation of breast cancer cells by competitive binding with estrogen. At the same time, the abnormalities of the abundance and / or activity of ERa co-regulators are often associated with the incidence and mortality of breast cancer, and are also potential targets for predicting and treating breast cancer. The C2H2 zinc finger protein family is the largest transcription/transcription regulatory factor family in mammals, whereas KRAB zinc finger protein only exists in quadruped vertebrates. It has the similar structure and mechanism of action, and with the rapid increase of species evolution. There are 423 kinds of zinc finger proteins in the human genome, accounting for about 60% of the total number of C2H2 zinc finger proteins. The KRAB domain of the N-terminal of KRAB type zinc finger proteins interacts with KAP1 to recruit transcriptional inhibitory proteins, forming transcriptional silencing complexes and exerting transcriptional inhibition functions. The C2H2 type zinc finger domain of the C-terminal mainly mediates specific DNA sequences or sequences. The binding of other transcription factors anchors the transcriptional silencing complex to specific chromatin sites. Increasing evidence suggests that the appearance and expansion of KRAB zinc finger proteins are closely related to the evolution of vertebrates and the establishment and improvement of their fine transcriptional regulatory networks. Normal physiological activities, such as cell proliferation and differentiation, are also associated with the occurrence and development of tumors. In general, the transcriptional regulatory network in cancer cells tends to simplify, and the abundance of KRAB-type zinc finger protein in cancer tissues is often lower than that in adjacent tissues. ZNF496 belongs to KRAB-type zinc finger protein and can be associated with NSD1 (nuclear rece 1) through its C2HR domain. Up to now, there is no detailed report about the function and mechanism of ZNF496. By analyzing the expression profile of ZNF496 on www.proteinatlas.org website, we found that ZNF496 is highly expressed in female reproductive system and breast, but rarely or not in the corresponding cancer tissues. Our results suggest that: 1. ZNF496 is highly expressed in the female mammary gland and reproductive system, but low in the corresponding cancer tissues. We purchased human reproductive system tumor tissue microarray and found that ZNF496 in breast cancer, ovarian cancer and ovarian cancer. The expression of ZNF496 in cervical cancer was lower than that in adjacent tissues. The relative expression of ZNF496 in the main tissues and organs of adult male and female rats was detected by WB. The results showed that ZNF496 was specifically expressed in lung, ovary, fallopian tube and uterus. The tissue-specific expression of ZNF496 may be related to the normal physiological function of these tissues and organs. 2. ZNF496 interacts with ERa. To explore the functional mechanism of ZNF496, we first identified ZNF496 as a potential interacting protein in MCF-7 nucleus by IP-MS. Mass spectrometry revealed that ERa may be a potential interacting protein in ZNF496. Subsequently, Co-IP confirmed that ZNF496 could interact with ERa regardless of E2 presence. Effect. 3. ZNF496 selectively inhibits the transcriptional activity of ERa: QPCR assay confirmed that the overexpression of ZNF496 could be inhibited under E2 treatment. Knocking down ZNF496 could promote the transcriptional level of ERa target genes Greb1, pS2, Xbp1, Sgk1 and Wisp2, but had no effect on the expression of Serpina 3, another target gene of ERa, and ZNF496 did not affect the mRNA and protein water of ERa. This indicates that ZNF496 can selectively regulate the transcriptional activity of ERalpha. 4. ZNF496 selectively inhibits the binding of ERalpha to some target gene promoters. Laboratory work has shown that the C2H2 zinc finger domain at the C-terminal of ZNF496 protein and the DBD domain in the middle of ERalpha protein mediate the interaction between them, suggesting that ZNF496 may affect the interaction between ERalpha and ER. In order to explore the mechanism of selective regulation of ERa by ZNF496, we conducted EMSA experiments. The results showed that the ability of ERa to bind to ERE sequence decreased gradually with the increase of expression of ZNF496 under E2 treatment. It was further suggested that the over-expression of ZNF496 could inhibit the binding of ERa to ERE sequence under E2 treatment. Finally, we detected the binding ability of endogenous ERa to the promoter region of its target gene in MCF-7 by Ch IP-q PCR. The results showed that after the over-expression of ZNF496 under E2 treatment, ERa binds to pS2, Greb1 and Wisp2 genes. ZNF496 could inhibit the proliferation of ER-positive breast cancer cells. Using lentiviral infection, the stable overexpression of ZNF496 cell lines MCF-7, T-47D and MDA-MB-231.CCK-8 were obtained. Cell proliferation assay showed that the overexpression of ZNF496 could be inhibited under E2 treatment. The proliferation of MCF-7 and T-47D had no effect on MDA-MB-231. The cloning formation assay also confirmed that the overexpression of ZNF496 could inhibit the cloning ability of MCF-7 and T-47D in normal medium, but had no effect on MDA-MB-231. Therefore, ZNF496 inhibited the proliferation of ER-positive breast cancer cells by selectively inhibiting the activity of ERa.6, ZNF496. Differential expression in breast cancer depends on the state of ER alpha: Immunohistochemical results from 29 breast cancer samples showed that the expression of ZNF496 was significantly lower in cancer tissues than in adjacent tissues. In conclusion, ZNF496 selectively inhibits the binding of ERa to ERA domain and decreases the transcriptional activity of ERA by competing with the domain of DBD binding to ERa. This study provides a new idea for further revealing the molecular mechanism of the occurrence and development of ER-alpha-positive breast cancer, provides a potential target for the diagnosis and treatment of breast cancer, and improves the understanding of the function and mechanism of KRAB-type zinc finger protein family members.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.9
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本文編號(hào)：2233757
[Abstract]:Estrogen receptor alpha (ERa), a member of the nuclear receptor superfamily, is involved in a variety of physiological and pathological processes. ERa, which is activated by estrogen binding, can form homologous dimers and bind to palindromic estrogen response elements (EREs) to activate transcription. The AF1 domain, AF2 domain and DBD domain are all transcription-activated domains, but only AF2 domain is estrogen-dependent. Abnormal endocrine levels are thought to be an important cause of breast cancer. About 3/4 of breast cancers are ER-alpha-positive, and excessive activation of ER-alpha can stimulate malignant transformation of normal cells, which is also a necessary condition for maintaining the proliferation of breast cancer cells. The changes of posttranslational modification can affect the signal pathway of estrogen receptor and participate in the occurrence and development of breast cancer to varying degrees. Therefore, ERa is a biomarker of breast cancer and is often used to guide the treatment of breast cancer. It can inhibit the growth and proliferation of breast cancer cells by competitive binding with estrogen. At the same time, the abnormalities of the abundance and / or activity of ERa co-regulators are often associated with the incidence and mortality of breast cancer, and are also potential targets for predicting and treating breast cancer. The C2H2 zinc finger protein family is the largest transcription/transcription regulatory factor family in mammals, whereas KRAB zinc finger protein only exists in quadruped vertebrates. It has the similar structure and mechanism of action, and with the rapid increase of species evolution. There are 423 kinds of zinc finger proteins in the human genome, accounting for about 60% of the total number of C2H2 zinc finger proteins. The KRAB domain of the N-terminal of KRAB type zinc finger proteins interacts with KAP1 to recruit transcriptional inhibitory proteins, forming transcriptional silencing complexes and exerting transcriptional inhibition functions. The C2H2 type zinc finger domain of the C-terminal mainly mediates specific DNA sequences or sequences. The binding of other transcription factors anchors the transcriptional silencing complex to specific chromatin sites. Increasing evidence suggests that the appearance and expansion of KRAB zinc finger proteins are closely related to the evolution of vertebrates and the establishment and improvement of their fine transcriptional regulatory networks. Normal physiological activities, such as cell proliferation and differentiation, are also associated with the occurrence and development of tumors. In general, the transcriptional regulatory network in cancer cells tends to simplify, and the abundance of KRAB-type zinc finger protein in cancer tissues is often lower than that in adjacent tissues. ZNF496 belongs to KRAB-type zinc finger protein and can be associated with NSD1 (nuclear rece 1) through its C2HR domain. Up to now, there is no detailed report about the function and mechanism of ZNF496. By analyzing the expression profile of ZNF496 on www.proteinatlas.org website, we found that ZNF496 is highly expressed in female reproductive system and breast, but rarely or not in the corresponding cancer tissues. Our results suggest that: 1. ZNF496 is highly expressed in the female mammary gland and reproductive system, but low in the corresponding cancer tissues. We purchased human reproductive system tumor tissue microarray and found that ZNF496 in breast cancer, ovarian cancer and ovarian cancer. The expression of ZNF496 in cervical cancer was lower than that in adjacent tissues. The relative expression of ZNF496 in the main tissues and organs of adult male and female rats was detected by WB. The results showed that ZNF496 was specifically expressed in lung, ovary, fallopian tube and uterus. The tissue-specific expression of ZNF496 may be related to the normal physiological function of these tissues and organs. 2. ZNF496 interacts with ERa. To explore the functional mechanism of ZNF496, we first identified ZNF496 as a potential interacting protein in MCF-7 nucleus by IP-MS. Mass spectrometry revealed that ERa may be a potential interacting protein in ZNF496. Subsequently, Co-IP confirmed that ZNF496 could interact with ERa regardless of E2 presence. Effect. 3. ZNF496 selectively inhibits the transcriptional activity of ERa: QPCR assay confirmed that the overexpression of ZNF496 could be inhibited under E2 treatment. Knocking down ZNF496 could promote the transcriptional level of ERa target genes Greb1, pS2, Xbp1, Sgk1 and Wisp2, but had no effect on the expression of Serpina 3, another target gene of ERa, and ZNF496 did not affect the mRNA and protein water of ERa. This indicates that ZNF496 can selectively regulate the transcriptional activity of ERalpha. 4. ZNF496 selectively inhibits the binding of ERalpha to some target gene promoters. Laboratory work has shown that the C2H2 zinc finger domain at the C-terminal of ZNF496 protein and the DBD domain in the middle of ERalpha protein mediate the interaction between them, suggesting that ZNF496 may affect the interaction between ERalpha and ER. In order to explore the mechanism of selective regulation of ERa by ZNF496, we conducted EMSA experiments. The results showed that the ability of ERa to bind to ERE sequence decreased gradually with the increase of expression of ZNF496 under E2 treatment. It was further suggested that the over-expression of ZNF496 could inhibit the binding of ERa to ERE sequence under E2 treatment. Finally, we detected the binding ability of endogenous ERa to the promoter region of its target gene in MCF-7 by Ch IP-q PCR. The results showed that after the over-expression of ZNF496 under E2 treatment, ERa binds to pS2, Greb1 and Wisp2 genes. ZNF496 could inhibit the proliferation of ER-positive breast cancer cells. Using lentiviral infection, the stable overexpression of ZNF496 cell lines MCF-7, T-47D and MDA-MB-231.CCK-8 were obtained. Cell proliferation assay showed that the overexpression of ZNF496 could be inhibited under E2 treatment. The proliferation of MCF-7 and T-47D had no effect on MDA-MB-231. The cloning formation assay also confirmed that the overexpression of ZNF496 could inhibit the cloning ability of MCF-7 and T-47D in normal medium, but had no effect on MDA-MB-231. Therefore, ZNF496 inhibited the proliferation of ER-positive breast cancer cells by selectively inhibiting the activity of ERa.6, ZNF496. Differential expression in breast cancer depends on the state of ER alpha: Immunohistochemical results from 29 breast cancer samples showed that the expression of ZNF496 was significantly lower in cancer tissues than in adjacent tissues. In conclusion, ZNF496 selectively inhibits the binding of ERa to ERA domain and decreases the transcriptional activity of ERA by competing with the domain of DBD binding to ERa. This study provides a new idea for further revealing the molecular mechanism of the occurrence and development of ER-alpha-positive breast cancer, provides a potential target for the diagnosis and treatment of breast cancer, and improves the understanding of the function and mechanism of KRAB-type zinc finger protein family members.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.9
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本文編號(hào)：2233757
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