ZNF496在乳腺癌發(fā)生發(fā)展中功能與作用機制的研究
發(fā)布時間：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,參與多種生理學和病理學過程。雌激素結合活化的ERα可形成同源二聚體,與回文的雌激素反應元件(estrogen response element,ERE)結合,發(fā)揮轉錄激活的功能。ERα主要包含3個重要的結構域,分別是AF1結構域、AF2結構域和DBD結構域。AF1和AF2都是轉錄激活結構域,但只有AF2結構域具有雌激素依賴性。DBD結構域主要介導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,具有十分復雜的發(fā)病機制。目前認為,人體內(nèi)分泌水平的異常是誘發(fā)乳腺癌的一個重要原因。約3/4的乳腺癌為ERα陽性,而ERα的過度活化能夠刺激正常細胞的惡性轉化,也是維持乳腺癌細胞增殖的一個必要條件。ERα與配體和共調(diào)節(jié)因子結合的失調(diào)以及自身結構和翻譯后化修飾的變化都會不同程度地影響雌激素受體的信號通路,參與乳腺癌的發(fā)生發(fā)展。因此,ERα是乳腺癌的生物標記物,常用于指導乳腺癌的治療。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的一個經(jīng)典拮抗劑,通過與雌激素競爭結合ERα,抑制ERα的活性,抑制乳腺癌細胞的生長和增殖。同時,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性,也是預測和治療乳腺癌的潛在靶標,因此對ERα調(diào)節(jié)蛋白的研究對揭示ERα的調(diào)控機制,鑒定乳腺癌的診斷和治療靶標具有重要的意義。C2H2型鋅指蛋白家族是哺乳動物中最大的轉錄/轉錄調(diào)控因子家族,而KRAB型鋅指蛋白只存在于四足脊椎動物中,具有相似的結構和作用機制,且隨著物種進化數(shù)量急劇增加,在人類基因組中數(shù)量高達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結構域通過與KAP1相互作用招募轉錄抑制蛋白,形成轉錄沉默復合物,發(fā)揮轉錄抑制的功能,而C端的C2H2型鋅指結構域主要介導與特定DNA序列或其他轉錄因子的結合,將轉錄沉默復合物錨定在特定染色質(zhì)位點。越來越多的證據(jù)表明,KRAB型鋅指蛋白的出現(xiàn)和數(shù)量膨脹與脊椎動物的進化及其精細轉錄調(diào)控網(wǎng)絡的建立和完善密切相關。KRAB型鋅指蛋白參與穩(wěn)定基因組結構、胚胎發(fā)育、細胞的增殖和分化等正常生理活動,又與腫瘤的發(fā)生發(fā)展有某些關聯(lián)。一般情況下,癌細胞中轉錄調(diào)控網(wǎng)絡趨于簡單化,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,可通過其C2HR結構域與NSD1(nuclear receptor binding SET domain protein 1)結合,介導KAP-1不依賴的轉錄抑制功能。目前,尚未有關于ZNF496功能及機制的詳細報道。通過www.proteinatlas.org網(wǎng)站對其表達譜分析,發(fā)現(xiàn)ZNF496在女性生殖系統(tǒng)和乳腺中高表達,但是在相應的癌組織中表達很少或不表達,提示ZNF496可能與乳腺癌的發(fā)生發(fā)展有關聯(lián),但是其中的機制尚不明確。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,在相應的癌組織中低表達。我們通過購買的人類生殖系統(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低于癌旁組織。我們還通過WB檢測了成年雄鼠和雌鼠的主要組織器官中ZNF496的相對表達豐度。結果顯示ZNF496在肺、卵巢、輸卵管和子宮中特異性表達。ZNF496的這種組織特異性表達可能與這些組織器官的正常生理功能的維持相關。2、ZNF496能夠與ERα相互作用:為了探討ZNF496的功能機制,我們首先通過IP-MS鑒定MCF-7細胞核中ZNF496的潛在相互作用蛋白,質(zhì)譜結果顯示ERα可能是ZNF496潛在的相互作用蛋白。隨后的Co-IP證實,無論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的轉錄活性:qPCR實驗證實,在E2處理條件下,ZNF496的過表達能夠抑制,敲低能夠促進ERα靶基因Greb1、pS2、Xbp1、Sgk1和Wisp2的轉錄水平,而對ERα另一個靶基因Serpina3的表達沒有影響,同時ZNF496并不影響ERα的mRNA和蛋白水平。這說明了ZNF496能夠選擇性調(diào)控ERα的轉錄活性。4、ZNF496選擇性抑制ERα與部分靶基因啟動子的結合:實驗室他人工作證實ZNF496蛋白C端的C2H2型鋅指結構域和ERα蛋白中部的DBD結構域介導了它們之間的相互作用,提示ZNF496可能影響ERα與ERE區(qū)的結合。熒光定位實驗證實,在E2處理條件下,ZNF496的過表達減弱活化的ERα在核內(nèi)的凝集現(xiàn)象。為了探討ZNF496選擇性調(diào)控ERα的機制,我們開展了EMSA實驗。結果顯示,在E2處理條件下,隨著ZNF496表達量的增加,ERα結合ERE序列的能力逐漸減弱。進一步提示,在E2處理條件下,ZNF496的過表達能夠抑制ERα與ERE序列的結合。最后我們利用Ch IP-qPCR實驗檢測MCF-7中內(nèi)源ERα與其靶基因啟動子區(qū)域的結合能力。結果顯示,在E2處理條件下,ZNF496過表達之后,ERα結合pS2、Greb1和Wisp2等基因啟動子的能力出現(xiàn)減弱趨勢,而ERα結合Serpina3啟動子的能力沒有變化。5、ZNF496可抑制ERα陽性乳腺癌細胞的增殖:利用慢病毒感染,獲得ZNF496穩(wěn)定過表達的細胞株MCF-7、T-47D和MDA-MB-231。CCK-8測細胞增殖實驗顯示,在E2處理條件下,ZNF496過表達能夠抑制MCF-7和T-47D的增殖,而對MDA-MB-231沒影響。克隆形成實驗也證實,在正常培養(yǎng)基培養(yǎng)條件下,ZNF496過表達能夠抑制MCF-7和T-47D的克隆形成能力,而對MDA-MB-231沒影響。因此,ZNF496通過選擇性抑制ERα的活性抑制ERα陽性乳腺癌細胞的增殖。6、ZNF496在乳腺癌中的差異表達依賴于ERα的狀態(tài):29例乳腺癌病例樣本的免疫組化結果顯示,ZNF496在癌組織中的表達顯著低于癌旁組織。當我們將乳腺癌樣本劃分為ERα+和ERα-時,ZNF496在ERα+乳腺癌組織中的表達極顯著低于癌旁組織,而ZNF496在ERα-乳腺癌組織中的表達與癌旁組織沒有顯著性差異。因此ZNF496在乳腺癌中的差異表達依賴于ERα的陽性表達。綜上所述,本研究發(fā)現(xiàn)ZNF496通過競爭結合ERα的DBD結構域選擇性抑制ERα與ERE區(qū)的結合,降低ERα的轉錄活性。ZNF496依賴對ERα的調(diào)控發(fā)揮抑制ERα陽性乳腺癌細胞增殖的作用。本研究為進一步揭示ERα陽性乳腺癌發(fā)生發(fā)展的分子機理提供了思路,為乳腺癌的診斷或治療提供了潛在靶標,同時完善了人們對KRAB型鋅指蛋白家族成員功能與作用機制的認識。
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9
，
本文編號：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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9
，
本文編號：2233757
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