ZNF496在乳腺癌发生发展中功能与作用机制的研究
发布时间：2018-09-10 07:14
【摘要】：雌激素受体α(estrogen receptor alpha,ERα)属于核受体超家族的一员,参与多种生理学和病理学过程。雌激素结合活化的ERα可形成同源二聚体,与回文的雌激素反应元件(estrogen response element,ERE)结合,发挥转录激活的功能。ERα主要包含3个重要的结构域,分别是AF1结构域、AF2结构域和DBD结构域。AF1和AF2都是转录激活结构域,但只有AF2结构域具有雌激素依赖性。DBD结构域主要介导ERα与DNA序列的相互作用。乳腺癌作为女性发病率最高的恶性肿瘤之一,具有十分复杂的发病机制。目前认为,人体内分泌水平的异常是诱发乳腺癌的一个重要原因。约3/4的乳腺癌为ERα阳性,而ERα的过度活化能够刺激正常细胞的恶性转化,也是维持乳腺癌细胞增殖的一个必要条件。ERα与配体和共调节因子结合的失调以及自身结构和翻译后化修饰的变化都会不同程度地影响雌激素受体的信号通路,参与乳腺癌的发生发展。因此,ERα是乳腺癌的生物标记物,常用于指导乳腺癌的治疗。目前,靶向ERα是乳腺癌辅助治疗中最常用的策略之一。Tamoxifen是ERα的一个经典拮抗剂,通过与雌激素竞争结合ERα,抑制ERα的活性,抑制乳腺癌细胞的生长和增殖。同时,ERα共调节因子的丰度和/或活性的异常往往与乳腺癌的发生率和致死率具有相关性,也是预测和治疗乳腺癌的潜在靶标,因此对ERα调节蛋白的研究对揭示ERα的调控机制,鉴定乳腺癌的诊断和治疗靶标具有重要的意义。C2H2型锌指蛋白家族是哺乳动物中最大的转录/转录调控因子家族,而KRAB型锌指蛋白只存在于四足脊椎动物中,具有相似的结构和作用机制,且随着物种进化数量急剧增加,在人类基因组中数量高达423种,占据了C2H2型锌指蛋白总数的60%左右。KRAB型锌指蛋白N端的KRAB结构域通过与KAP1相互作用招募转录抑制蛋白,形成转录沉默复合物,发挥转录抑制的功能,而C端的C2H2型锌指结构域主要介导与特定DNA序列或其他转录因子的结合,将转录沉默复合物锚定在特定染色质位点。越来越多的证据表明,KRAB型锌指蛋白的出现和数量膨胀与脊椎动物的进化及其精细转录调控网络的建立和完善密切相关。KRAB型锌指蛋白参与稳定基因组结构、胚胎发育、细胞的增殖和分化等正常生理活动,又与肿瘤的发生发展有某些关联。一般情况下,癌细胞中转录调控网络趋于简单化,而癌组织中KRAB型锌指蛋白的丰度也往往低于癌旁组织。ZNF496属于KRAB型锌指蛋白,可通过其C2HR结构域与NSD1(nuclear receptor binding SET domain protein 1)结合,介导KAP-1不依赖的转录抑制功能。目前,尚未有关于ZNF496功能及机制的详细报道。通过www.proteinatlas.org网站对其表达谱分析,发现ZNF496在女性生殖系统和乳腺中高表达,但是在相应的癌组织中表达很少或不表达,提示ZNF496可能与乳腺癌的发生发展有关联,但是其中的机制尚不明确。我们的研究发现:1、ZNF496在女性乳腺和生殖系统中高表达,在相应的癌组织中低表达。我们通过购买的人类生殖系统肿瘤组织芯片发现,ZNF496在乳腺癌、卵巢癌和宫颈癌中的表达低于癌旁组织。我们还通过WB检测了成年雄鼠和雌鼠的主要组织器官中ZNF496的相对表达丰度。结果显示ZNF496在肺、卵巢、输卵管和子宫中特异性表达。ZNF496的这种组织特异性表达可能与这些组织器官的正常生理功能的维持相关。2、ZNF496能够与ERα相互作用:为了探讨ZNF496的功能机制,我们首先通过IP-MS鉴定MCF-7细胞核中ZNF496的潜在相互作用蛋白,质谱结果显示ERα可能是ZNF496潜在的相互作用蛋白。随后的Co-IP证实,无论E2是否存在,ZNF496均能够与ERα发生相互作用。3、ZNF496选择性抑制ERα的转录活性:qPCR实验证实,在E2处理条件下,ZNF496的过表达能够抑制,敲低能够促进ERα靶基因Greb1、pS2、Xbp1、Sgk1和Wisp2的转录水平,而对ERα另一个靶基因Serpina3的表达没有影响,同时ZNF496并不影响ERα的mRNA和蛋白水平。这说明了ZNF496能够选择性调控ERα的转录活性。4、ZNF496选择性抑制ERα与部分靶基因启动子的结合:实验室他人工作证实ZNF496蛋白C端的C2H2型锌指结构域和ERα蛋白中部的DBD结构域介导了它们之间的相互作用,提示ZNF496可能影响ERα与ERE区的结合。荧光定位实验证实,在E2处理条件下,ZNF496的过表达减弱活化的ERα在核内的凝集现象。为了探讨ZNF496选择性调控ERα的机制,我们开展了EMSA实验。结果显示,在E2处理条件下,随着ZNF496表达量的增加,ERα结合ERE序列的能力逐渐减弱。进一步提示,在E2处理条件下,ZNF496的过表达能够抑制ERα与ERE序列的结合。最后我们利用Ch IP-qPCR实验检测MCF-7中内源ERα与其靶基因启动子区域的结合能力。结果显示,在E2处理条件下,ZNF496过表达之后,ERα结合pS2、Greb1和Wisp2等基因启动子的能力出现减弱趋势,而ERα结合Serpina3启动子的能力没有变化。5、ZNF496可抑制ERα阳性乳腺癌细胞的增殖:利用慢病毒感染,获得ZNF496稳定过表达的细胞株MCF-7、T-47D和MDA-MB-231。CCK-8测细胞增殖实验显示,在E2处理条件下,ZNF496过表达能够抑制MCF-7和T-47D的增殖,而对MDA-MB-231没影响。克隆形成实验也证实,在正常培养基培养条件下,ZNF496过表达能够抑制MCF-7和T-47D的克隆形成能力,而对MDA-MB-231没影响。因此,ZNF496通过选择性抑制ERα的活性抑制ERα阳性乳腺癌细胞的增殖。6、ZNF496在乳腺癌中的差异表达依赖于ERα的状态:29例乳腺癌病例样本的免疫组化结果显示,ZNF496在癌组织中的表达显著低于癌旁组织。当我们将乳腺癌样本划分为ERα+和ERα-时,ZNF496在ERα+乳腺癌组织中的表达极显著低于癌旁组织,而ZNF496在ERα-乳腺癌组织中的表达与癌旁组织没有显著性差异。因此ZNF496在乳腺癌中的差异表达依赖于ERα的阳性表达。综上所述,本研究发现ZNF496通过竞争结合ERα的DBD结构域选择性抑制ERα与ERE区的结合,降低ERα的转录活性。ZNF496依赖对ERα的调控发挥抑制ERα阳性乳腺癌细胞增殖的作用。本研究为进一步揭示ERα阳性乳腺癌发生发展的分子机理提供了思路,为乳腺癌的诊断或治疗提供了潜在靶标,同时完善了人们对KRAB型锌指蛋白家族成员功能与作用机制的认识。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
，
本文编号：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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
，
本文编号：2233757
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2233757.html
