miR-155/PU.1/TAL-1通路调控乳腺癌细胞生物学功能的机制研究

miR-155 is closely related with the occurrence and development of breast cancer, but the mechanism is still unclear.In the preliminary work, we have recognized a specific regulatory pathway miR-155/PU.1/TAL-1 of breast caner using the bioinformatics methods. And using biological experiments, we have found that miR-155 target PU.1 and miR-155 overexpression can decrease PU.1 expression but increase TAL-1 expression. And RNA interference silencing of TAL-1 inhibits proliferation and promotes apoptosis of breast cancer cells. Thus, we speculated that in breast cancer, miR-155 exerts its function by negatively regulating PU.1 and further increasing TAL-1 expression. In this study, we will confirm miR-155 targeting PU.1 by luciferase and verify PU.1 targeting TAL-1 with ChIP technology. In the breast cancer cells, we will observe the expression of target genes and the biological behavior variation in cells after miR-155, PU.1 and TAL-1 are knocked down or overexpressed using siRAN technology or cell transfection, respectively; In the case-control breast cancer tissues, we will observe the expression of miR-155, PU.1 and TAL-1 and their correlations with clinical significance using real-time PCR and Western Blot technology. Our project will prove that miR-155/PU.1/TAL-1 pathway is a new mechanism of miR-155 in breast cancer from cellular and histological levels. And the results will provide new ideas and targets for breast cancer prevention and treatment.

miR-155与乳腺癌的发生发展密切相关，但其作用机制尚未完全阐明。我们前期工作用生物信息学方法预测出乳腺癌特异的调控通路miR-155/PU.1/TAL-1，并证实在乳腺癌细胞中miR-155靶向PU.1，miR-155过表达使PU.1降低、TAL-1增加，沉默TAL-1能够抑制细胞增殖、促进凋亡。由此推测在乳腺癌中miR-155通过抑制PU.1进而上调TAL-1的表达发挥作用。本项目拟通过ChIP检测PU.1与TAL-1的靶向关系，在乳腺癌细胞中通过细胞转染和siRNA技术将miR-155、PU.1和TAL-1分别过表达或沉默，观察对下游靶基因及细胞的生物学功能的影响；在配对乳腺癌组织中用荧光定量PCR和Western Blot观察miR-155、PU.1和TAL-1表达量及与临床病理的相关性。从细胞和组织水平证明该通路是miR-155发挥作用的新机制，为乳腺癌防治提供新思路和新靶点。

本项目按计划完成，并进行了相应的扩展研究。miR-155与乳腺癌的发生发展密切相关，但其作用机制尚未完全阐明。本课题将生物信息学方法与生物学实验相结合，探讨miR-155在乳腺癌中发挥作用的新机制。首先，课题组利用生物信息学方法整合了TransFAC、TransmiR、TarBase和miRecords四个数据库的人类转录和后转录调控关系数据，构建背景调控网络，并从Gene Expression Omnibus和ArrayExpress数据库中获取了5套乳腺癌相关的基因芯片数据，利用meta分析最终识别出得分最高的miR-155/PU.1/TAL-1调控通路。然后，我们根据预测的结果对该调控通路进行证实和深入研究，取得了以下进展：通过双荧光素酶报告基因分析实验证实，在乳腺癌细胞中miR-155能够靶向PU.1；在细胞水平上，乳腺癌细胞中过表达miR-155使 PU.1 降低，反之，敲低miR-155使PU.1表达上调，无论过表达或干扰miR-155或PU.1都能够影响乳腺癌细胞的生物学功能；在组织水平上，用荧光定量PCR和免疫组化方法检测乳腺癌组织和配对的癌旁对照乳腺组织中miR-155和PU.1的表达，未发现两者的相关性，但我们发现不仅miR-155在癌组织中高表达，PU.1在癌组织中的表达也高于对照乳腺组织，而且PU.1的表达与乳腺癌Her-2表达呈正相关。但是遗憾的是无论在乳腺癌细胞系和乳腺癌样本中都检测不到TAL-1的表达。本课题从新的视角阐明了miR-155发挥作用的新机制，为乳腺癌防治提供新思路和新靶点。项目在研期间已发表署名本课题资助的SCI论文2篇，投稿1篇。

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