【作者】 冯旭；

【导师】 邓务国；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 目的:乳腺癌是全球癌症中导致女性死亡的主要因素之一。目前主要的治疗方法包括手术、放疗以及化疗。然而,即使通过治疗,乳腺癌也存在一定的可能性复发,而造成这种可能的主要原因之一,便是由于乳腺癌干细胞的存在。研究表明,乳腺癌组织中存在一群CD44+CD24-的肿瘤细胞,它们具有极强的复制及自我更新能力,将此细胞群称为乳腺癌干细胞。它们数量极少,但对于常规的放化疗不敏感,因此靶向乳腺癌干细胞是目前治疗乳腺癌的重要方向。NMI是一种能与多种转录因子结合的蛋白,它通过与其它转录因子的结合,参与多种细胞活动,如DNA损伤、细胞周期及上皮-间质转化等,并且在乳腺癌细胞中,主要发挥抑癌作用。然而,NMI对于乳腺癌干细胞的影响目前知之甚少。因此,进一步研NMI在乳腺癌干细胞发生发展中的功能和具体的分子机制,从而为乳腺癌的诊断和治疗提供新靶标具有重要意义。方法:1.通过成球实验富集乳腺癌干细胞并利用流式细胞术检测其CD44/CD24表达情况;利用Western blot及q RT-PCR检测NMI及干性指标Nanog、Oct4及Sox2的表达情况;利用GEO数据库GSE15192 m RNA表达数据,分析在CD44+CD24-及CD44-CD24+细胞群体中,NMI、CD44及CD24的表达情况。2.敲低及过表达NMI后,通过Western blot,q RT-PCR,成球实验及流式细胞术,检测NMI对于乳腺癌干细胞干性特性的影响。3.通过检测NMI敲低后,h TERT启动子荧光素酶活性,检测NMI是否能影响h TERT转录活性;通过检测NMI敲低后进行Western blot,检测NMI能否影响h TERT蛋白水平;通过敲低NMI同时敲低h TERT,或者过表达NMI同时过表达h TERT后进行Western blot、流式细胞术、成球实验、transwell及免疫荧光,检测NMI对于乳腺癌细胞干性特点的影响,是否是通过h TERT介导的。4.动物实验,通过皮下注射肿瘤细胞,构建裸鼠荷瘤动物模型,检测NMI稳定敲低/过表达的细胞系及其对照细胞系成瘤能力变化情况,并同时引入h TERT,进行rescue实验;通过尾静脉注射NMI稳定过表达细胞系及其对照细胞系,检测NMI过表达后,对于乳腺癌细胞转移能力的影响。5.利用GEO数据库,分析NMI与h TERT表达的相关性;利用组织芯片,进行免疫组织化学染色,检测NMI与h TERT表达的相关性;分析NMI,h TERT与临床病理分期及预后的关系。6.利用免疫共沉淀及质谱,寻找与NMI共同作用于h TERT的转录因子,并进行Western blot验证。结果:1.相比于乳腺癌细胞,NMI在乳腺癌干细胞中呈低表达;2.敲低NMI,能增强干性相关基因NANOG、OCT4及SOX2的表达,增强乳腺癌细胞成球能力及CD44+CD24-细胞群体比例;3.过表达NMI,能抑制干性相关基因NANOG、OCT4及SOX2的表达,抑制乳腺癌细胞成球能力及CD44+CD24-细胞群体比例;4.NMI通过抑制h TERT转录,从而影响乳腺癌细胞干性特征;5.NMI通过h TERT影响乳腺癌细胞上皮-间质转化能力及裸鼠成瘤能力;6.在临床样本中,NMI与h TERT表达呈负相关;7.NMI通过与YY1相互作用下调h TERT。结论:在正常乳腺组织中NMI表达水平较癌组织高,且NMI与h TERT表达呈负相关。细胞水平实验表明,NMI能抑制乳腺癌细胞干性特点,且该能力是由h TERT介导。NMI通过抑制h TERT转录,从而抑制乳腺癌细胞干性特点。更多还原

【Abstract】 Objective: Breast cancer is one of the leading causes of cancer death among women worldwide.The main therapeutic approaches to treat breast cancer include surgery,radiotherapy and chemotherapy.However,it has some chances to recurrence and metastasis.Cancer stem cells(CSCs),including breast cancer stem cells(BCSCs),have been shown to play important roles in cancer’s recurrence and metastasis.BCSCs are also relatively resistant to chemotherapy and radiotherapy compared with their non-tumorigenic progeny.Therefore,it is necessary to discover and identify the novel and specific molecular targets or related signaling pathways to inhibit the mammary stem/progenitor cell population and suppress carcinogenesis and tumor metastasis.N-myc and STAT interactor(NMI)is such a protein that involved in a variety of signaling mechanisms and interacts with different transcription factors.It is associated with DNA damage,cell cycle control and epithelial-mesenchymal transition.However the role of NMI in cancer stem cell remains poorly understood.In this study,we try to explore the function of NMI in BCSCs and find out the mechanisms.Methods: 1.Mammosphere formation assay was used to detect the expression level of NMI in breast cancer stem cell enriched populations.Breast cancer stem cell surface markers(CD44/CD24)were used to detect the effect of NMI to breast cancer stem cells by flow cotymetry.q RT-PCR and Western blot were used to evaluate the expression level of NMI and stem cell associated proteins Nanog,Oct4 and Sox2.TheNMI m RNA levels in CD44+CD24-and CD44-CD24+ cell populations were analyzed using GEO database(GSE15192).2.Mammosphere formation ability,flow cytometry,western blot and q RT–PCR were used to detected the influence of NMI in breast cancer stem cells by knockdown/overexpression of NMI.3.Dual Luciferase Assays of h TERT promoter were used in MCF7 cells transfected with NMI knockdown plasmids.Western blot was used to detect the effect of NMI on the level of h TERT protein after NMI knockdown.Western blot,flow cytometry,mammosphere formation,transwell and immunofluorescence were used to detect the effect of NMI if it can be rescued by h TERT.4.Animal experiment was performed by subcutaneous injection of tumor cells to construct nude mice tumor model.The Tumor formation ability of NMI stable knockdown or overexpression cell lines and their control cell lines were detected.Intravenous injection was performed to construct breast cancer lung metastasis model.5.GEO database and immunohistochemistry were used to analyze the relationship between NMI and h TERT.The relationship between NMI/h TERT and clinic pathological stage were analyzed.6.Immunoprecipitation and mass spectrometry were used to find the transcription factor cooperated with NMI on h TERT promoter and verified by Western blot.Results: 1.NMI is downregulated in breast cancer stem cell(BCSC)-enriched populations.2.NMI knockdown promotes the expression of CSC-related markers NANOG,OCT4,SOX2,mammosphere formation ability and CD44+CD24-cells population.3.NMI overexpression inhibits the expression of CSC-related markers Nanog,Oct4,Sox2,mammosphere formation ability and CD44+CD24-cells population.4.NMI inhibits BCSCs traits by down-regulating h TERT expression.5.NMI suppresses EMT in breast cancer cells and tumorigenicity in mouse model with xenografts of human breast cancer by down-regulating h TERT.6.NMI inversely correlates with h TERT in breast cancer samples.7.YY1 interacts with NMI to mediate the down-regulation of h TERT.Conclusion: Our findings show that NMI was lower expressed in tumor tissue compared with normal tissue and NMI inhibits cancer stem-like cell traits in breast cancer by downregulation of h TERT.更多还原