切割及多聚腺苷酸化特异因子CPSF4调控乳腺癌转移特性的分子机制研究

Metastasis is the leading cause for breast cancer treatment failure. We carried out immunohistochemistry staining of CPSF4 and found that high expression of CPSF4 was correlated with more positive lymph node metastases and distant metastases in breast cancer. In vitro studies, CPSF4 was more highly expressed in high metastatic potential cell lines MDA-MB-231 than in low metastatic potential lines MCF-7. Overexpression of CPSF4 in MCF-7 cells caused a significant decrease in the expression of epithelial marker, whereas expression of mesenchymal markers was elevated. Knockdown of CPSF4 caused a significant decrease in cell migration and decrease in the expression of mesenchymal markers compared to nagative control, whereas epithelial marker increased. CPSF4 expression was significantly upregulated in metastatic breast cancer samples and associated with shorter five years survival of TCGA dataset by using bioinformatics analysis. However, the mechanism of CPSF4-mediated breast tumor metastases remains unclear. In this study, we will adopt technology such as RNAi, TALEN, SSH, and ChIP investigating molecular mechanism governing metastasis characteristics in breast cancer regulated by CPSF4, and analysize the association between CPSF4 and breast cancer recurrence, metastasis and survival. We hope our study will help us find new molecular markers and therapeutic targets for breast cancer.

转移是乳腺癌治疗失败的主要原因。我们前期工作发现，转录调节因子CPSF4高表达的乳腺癌淋巴结转移率和远处转移率高；体外实验显示CPSF4在高转移能力细胞株MDA-MB-231相比于低转移能力细胞株MCF-7表达上调，外源性过表达CPSF4促进MCF-7细胞的EMT表型，用siRNA抑制CPSF4表达可逆转MDA-MB-231细胞的EMT表型；通过生物信息学分析发现，CPSF4在转移性乳腺癌中表达显著高于非转移性乳腺癌，高表达的CPSF4乳腺癌5年生存率低。然而，CPSF4调控乳腺癌转移特性的分子机制尚不明确。本课题将采用RNA干扰、TALEN、抑制消减杂交、ChIP等技术，利用人乳腺癌细胞、动物模型和人体样本，研究CPSF4调控乳腺癌转移特性的分子机制及其与乳腺癌复发、转移和生存的关系，为确立CPSF4作为新型的乳腺癌分子标记和治疗靶点提供充分的理论基础和实验依据。

三阴型乳腺癌被定义为激素受体以及人表皮生长因子受体-2（HER-2）阴性的乳腺癌，转移是三阴型乳腺癌患者死亡的主要原因，然而，转移性三阴型乳腺癌至今缺乏非常有效的治疗措施。CPSF4（切割和多聚腺苷酸化特异因子4）是真核生物3’端poly（A）尾的加工元件之一。据报道，CPSF4可作为“转录共激活因子”参与基因转录过程，但其在三阴型乳腺癌中的功能至今尚无报道。功能实验发现CPSF4可促进三阴乳腺癌细胞侵袭、迁移以及裸鼠转移瘤形成，同时发现CPSF4可诱导上皮-间质转化发生，深入探索其分子机制发现CPSF4可结合于MDM4启动子区并上调其启动子活性促进其转录。MDM4（鼠双微体4）是p53的关键负性调控因子。回复实验证实了CPSF4调控三阴乳腺癌细胞转移的功能依赖于MDM4。临床标本分析提示CPSF4/MDM4高表达的乳腺癌患者预后较差，且CPSF4的表达与MDM4的表达呈正相关。另外，我们发现CPSF4参与RNA可变剪切过程。总的来说，本项目深入研究了CPSF4/MDM4在乳腺癌中的生物学功能、分子机制和临床转化价值，为肿瘤的诊治提供了新的靶点和理论支持。