DACH1通过YB-1抑制KLF5转录因子在三阴性乳腺癌 表达的功能和机制研究

KIZ OpenIR > 科研部门 > 肿瘤生物学（陈策实）

	DACH1通过YB-1抑制KLF5转录因子在三阴性乳腺癌表达的功能和机制研究
其他题名	DACH1 suppresses the expression of KLF5transcription factor in triple negative breast cancer via YB-1
	李思源
学位类型	硕士
导师	陈策实
	2016-07
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	Klf5 Dach1 Yb-1 三阴性乳腺癌
其他摘要	乳腺癌是严重危害女性身心健康的恶性肿瘤，在我国，乳腺癌的发病率和死亡率都在逐年上升，并且呈现有年轻化的趋势。近年来虽然在乳腺癌的诊断、治疗水平方面有了很大提高，但由于乳腺癌本身的异质性，在治疗乳腺癌过程中仍然遇到了一些困难。三阴性乳腺癌（TNBC）是雌激素受体（ERa）、孕激素受体（PR）、人类表皮生长因子受体（Her2）均为阴性的乳腺癌亚型，在乳腺癌患者中大约占10-15%，患者预后极差，且生存时间较短，对内分泌治疗不敏感，因而目前尚缺乏有效的靶向治疗手段。KLF5是一个重要的转录因子，其异常表达与结直肠癌、乳腺癌、膀胱癌和前列腺癌等的发生、发展密切相关。我们前期的研究发现，转录因子KLF5在TNBC中高表达，并且可以促进TNBC细胞的增殖、生长、迁移以及维持乳腺肿瘤干细胞干性。KLF5的高表达，预示着乳腺癌患者的预后较差，是TNBC有效的潜在治疗靶点。文献报道KLF5在转录水平受到RARα、NFkB、b-catenin、EGR1等的调节，但是其在三阴性乳腺癌的具体转录调控机制仍需要进一步探索。细胞命运决定因子DACH1是一个与果蝇视网膜及四肢发育相关的基因，并且在很多癌症中包括乳腺癌中表现出异常低表达，长期以来扮演着抑癌基因的角色，但DACH1在三阴性乳腺癌中的功能和具体机制目前还没有被详细报道。我们研究发现，细胞命运决定因子DACH1在TNBC细胞中可能部分通过抑制KLF5的功能，抑制细胞的增殖、生存和侵袭。DACH1的抑癌功能可能部分通过抑制转录因子YB-1，从而下调KLF5及其下游靶基因的表达。我们得出一个结论：DACH1可能通过YB-1抑制KLF5及其下游靶基因的表达，从而抑制三阴性乳腺癌。研究中我们首先明确了DACH1和YB-1在体内、体外调节TNBC的功能。我们在乳腺上皮细胞系以及三阴性乳腺癌细胞系中检测了DACH1、YB-1以及KLF5的表达情况，发现DACH1与YB-1/KLF5在TNBC细胞系中的蛋白表达水平正好相反DACH1在TNBC中低表达，而YB-1和KLF5在TNBC均呈现高表达。接下来，我们在两株三阴性乳腺癌细胞系HCC1937和HCC1806中过表达DACH1，发现DACH1可以抑制的细胞增殖、DNA合成以及侵袭，并促进细胞凋亡。我们发现DACH1抑制KLF5表达，为了解析DACH1通过何种分子机制抑制KLF5，我们在HEK293细胞中过量表达DACH1以及YB1，观察到内源KLF5会被YB-1上升，DACH1则抑制YB-1这种功能。此外，我们单独在HCC1937和HCC1806中敲低DACH1以及YB-1, 发现敲低YB-1导致内源的KLF5下降, 敲低DACH1引起KLF5表达水平上升。Luciferase报告基因实验表明，YB-1可以促进KLF5启动子活性，而DACH1可以降低KLF5启动子的活性。Oligo pull-down实验证明，YB-1可以直接结合到KLF5启动子区域的CCAAT序列。这些结果组合在一起初步说明DACH1降低KLF5表达是在转录水平，DACH1可能通过抑制YB-1调控KLF5表达。综上所述，这些研究表明了，DACH1、YB-1和KLF5在三阴性乳腺癌中异常表达，DACH1降低KLF5的表达发生在转录水平，并且可能部分通过YB-1介导，从而抑制KLF5转录，抑制三阴性乳腺癌细胞的增殖、生存和侵袭。DACH1、YB-1和KLF5组合在一起可能是潜在的三阴性乳腺癌预后标志物或治疗靶标。本研究可为将来TNBC的临床治疗提供新的策略。; Breast cancer is one of the most malignant tumors, which harm to women's physical and mental health. In China, morbidity and mortality of breast cancer are on the rise year by year. In recent years, although the diagnosis and treatment of breast cancer had significant progress, breast cancer could not be cured due to its heterogeneity. ER, PR and Her2 Triple negative breast cancer (TNBC) is one of breast cancer subtypes that account for about 10-15%. The prognosis of TNBC patients is poor. In addition, patients are not sensitive to endocrine therapy. Currently, TNBC is lack of effective targeted therapies.KLF5 is an important transcription factor, whose abnormal expression is closely related with colorectal cancer, breast cancer, bladder cancer and prostate cancer. In our previous studies, we found that the KLF5 transcription factor high expressed in TNBC. KLF5 promotes TNBC cell proliferation, migration and stemness. KLF5 is an effective therapeutic target in TNBC. The transcription of KLF5 is regulated by RARa, NFkB, b-catenin and EGR1, but the regulation mechanism of KLF5 in TNBC needs to be further explored.Cell fate determination factor DACH1 is a gene related to drosophila retinal development. In many cancers including breast cancer, DACH1 is lowly expressed, suggesting that DACH1 plays a tumor suppressor role. However, the functions and mechanisms of DACH1 in TNBC have not been well studied.In this study, we found that DACH1 inhibits the KLF5 expression, cell proliferation and invasion and promotes apoptosis in TNBC cells. We found that DACH1 may inhibit KLF5 and its downstream target genes expression, inhibits cell proliferation and inducing apoptosis through inhibiting the transcription factor YB-1.We first defined the functions of DACH1 and YB-1 in TNBC. We detected the expression of DACH1, KLF5 and YB-1 in mammary epithelial cell lines and TNBC cell lines and found that the protein expression levels of DACH1 and KLF5/YB-1 are the opposite. DACH1 is lowly expressed but KLF5 and YB-1 are highly expressed in TNBC. Next, we overexpressed DACH1 in two TNBC cell lines HCC1937 and HCC1806 and found that overexpression of DACH1 can inhibit the expression of KLF5 and the cell proliferation and invasion and promoting apoptosis. Then we demonstrated that the molecular mechanism by which DACH1 inhibits KLF5 is mediated by YB-1. Firstly, we overexpressed YB-1 or DACH1 in HEK293 cell line and observed that endogenous KLF5 expression levels were increased by YB-1 and inhibited by DACH1; Secondly, we knocked down DACH1 and YB-1 and observed that DACH1 depletion increased and YB-1 depletion decreased the KLF5 expression levels. The Luciferase report assay showed that DACH1 inhibited and YB-1 increased the KLF5 promoter activity. Oligo pull-down experiments showed that YB-1 can bind to the CCAAT Box located at the KLF5 gene proximal promoter. These results suggest that DACH1 inhibits the KLF5 expression is at the transcriptional level and DACH1 functions through YB-1.Taken together, we showed that the DACH1, YB-1 and KLF5 are abnormal expressed in TNBC. DACH1 inhibits the KLF5 transcription partially by suppressing the YB-1 transactivation. DACH1 inhibits TNBC cell proliferation, survival and invasion through KLF5. DACH1, YB-1 and KLF5 together may be potential prognosis biomarkers or therapeutic targets in TNBC. This study may provide new strategies for clinical treatment in TNBC
学科领域	细胞生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/11960
专题	科研部门_肿瘤生物学（陈策实）
作者单位	中国科学院昆明动物研究所
推荐引用方式 GB/T 7714	李思源. DACH1通过YB-1抑制KLF5转录因子在三阴性乳腺癌表达的功能和机制研究[D]. 北京. 中国科学院研究生院,2016.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
201328010415024.pdf（3479KB）	学位论文		开放获取	CC BY-NC-SA	请求全文