中国科学院昆明动物研究所机构知识库

乳腺癌根据基因表达的情况可以被分成四种亚型：luminal A, luminal B，HER2过表达以及三阴型乳腺癌。其中三阴性乳腺癌患者的预后最差，并且目前缺乏有效的靶向治疗手段。YB-1属于冷激蛋白家族成员，主要作为转录因子结合到一系列靶基因的启动子区域发挥转录调节功能。同时,YB-1是胞浆中mRNPs的重要组分，与mRNA的翻译调控密切相关。YB-1高表达的乳腺癌患者的肿瘤恶性程度、复发几率都比YB-1低表达的患者高。根据文献报道，DACH1是YB-1的转录共抑制因子，能够抑制YB-1对KLF4的转录，鉴于KLF5与KLF4具有高度的同源性，以及KLF5和YB-1在乳腺癌的发生发展中的重要性，我们推测YB-1也能够调控KLF5转录，进而调控乳腺癌细胞的功能。本文首先对TCGA数据库进行分析，发现在乳腺癌样本中，KLF5表达与YB-1表达呈正相关，而与DACH1表达呈负相关。在HCC1937和HCC1806两株三阴性乳腺癌细胞中用siRNA沉默YB-1的表达，KLF5及其下游靶基因FGF-BP和CyclinD1的表达在mRNA和蛋白水平都随之受到下调。通过染色质免疫共沉淀实验，我们发现YB-1能够结合到KLF5启动子上。通过荧光素酶报告基因实验，我们验证了YB-1对KLF5启动子的活化依赖于KLF5启动子上的CCAAT box。同时，我们发现DACH1也能够结合到KLF5启动子上，并且依赖于YB-1。在HCC1937和HCC1806细胞中稳定敲低YB-1能够显著抑制细胞的生长。通过回复实验进一步发现YB-1促进三阴性乳腺癌细胞的增殖部分通过KLF5介导。我们通过裸鼠成瘤实验验证了在体内环境条件下，YB-1能够促进肿瘤生长，并且通过回复实验验证了YB-1促进肿瘤生长部分通过KLF5介导。根据本文研究，我们得出以下结论：在乳腺癌中KLF5表达与YB-1表达呈正相关，而与DACH1表达呈负相关。KLF5是YB-1的直接靶基因，YB-1通过结合到KLF5启动子上的CCAAT box激活KLF5转录。作为YB-1的转录共抑制因子，DACH1能够通过YB-1结合到KLF5启动子上。在细胞功能上，YB-1部分通过KLF5促进三阴性乳腺癌细胞和肿瘤生长。 

As one of the most common malignancies among women, breast tumors can be classified into the four subcategories: luminal A, luminal B, HER2-enriched and triple-negative subtypes according to the gene expression profiles. The triple negative breast cancer (TNBC) has the poorest prognosis and does not have effective targeted therapeutics because of lacking specific molecular targets.Y-box binding protein 1(YB-1) belongs to the cold shock protein superfamily and function as a transcription factor in the nucleus via binding to the promoter region of target genes.Meanwhile, YB-1was the part of mRNPs and regulated mRNA translation. Breast cancer with high YB-1 expression was more aggressive than YB-1 negative breast cancer and the recurrent risk was higher for the breast cancer patients with high YB-1 expression. DACH1 had been reported to be a co-inhibitor of YB-1 and inhibited the transcriptional activation of KLF4 by YB-1. There is great similarity between KLF5 and KLF4 because both YB-1 and KLF5 play important roles in breast cancer progression. We hypothesized that YB-1 promtoes KLF5 transcription and breast cancer cell proliferation.We first analyzed the relationship between the expression of DACH1, YB-1, and KLF5 in TCGA database and found that KLF5 expression is positively correlated with YB-1, but negatively correlated with DACH1.We knocked down YB-1 expression by siRNA in two TNBC cell lines and we found that the expression of KLF5 and its downstream targets, FGF-BP and CyclinD1, were reduced at both mRNA and protein levels. Using ChIP assays, we found that YB-1could bind toKLF5 promoter region. According to the results of dual luciferase reporter assays, we found that YB-1 activated KLF5 promoter activity, which is dependent on the CCAAT box in the KLF5 promoter. We also demonstrated that DACH1 bound to KLF5 promoter depending on YB-1. When YB-1 was stably knocked down in HCC1937 and HCC1806 cells, the cell viability and proliferation was significantly inhibited. Furthermore, KLF5 overexpression partially rescued the YB-1 knockdown induced cell viability and cell proliferation inhibition. Finally, we performed xenograft assays using YB-1 stably knockdown HCC1806 cells and found that silencing YB-1 expression significantly reduced tumor growth in nude mice and ectopic overexpression of KLF5 could partially rescue YB-1 depletion-induced tumor growth inhibition.In summary, we demonstrated that KLF5 expression is positively correlated with YB-1 expression, but negatively correlated with DACH1 expression in breast cancer. KLF5 is a direct transcriptional target of YB-1. YB1 binds to a CCAAT box at the KLF5 gene promoter and promotes KLF5 transcription. DACH1, which is the co-inhibitor of YB-1, can also bind to KLF5 promoter, depending on YB-1. Functionally, YB-1 promotes TNBC cell proliferation and tumor growth partially via regulating KLF5 expression.