RNA结合蛋白POP7/RPP20促进乳腺癌发生发展的功能及机制研究

Elucidation of the molecular mechanisms underlying breast cancer development and progression and identification of new therapeutic targets for this complex disease are two of key scientific questions to be addressed in the field of breast cancer research. Ribonuclease P protein subunit P20 (RPP20), encoded by processing of precursor 7, ribonuclease P/MRP subunit (POP7) gene, is one of protein subunits of the ribonuclease P/MRP complex. To date, the biological functions and related mechanisms for POP7/RPP20 in cancer development and progression remain unexplored. Recently, we identified POP7/RPP20 is a potential breast cancer oncoprotein through RNA-sequencing and CRISPR/Cas9 knockout screening using the well-recognized breast cancer progression model including immortalized human normal breast epithelial MCF10A, pre-malignant MCF10AT, comedo ductal carcinoma in situ MCF10DCIS.com, and metastatic MCF10CA1a cell lines. Kaplan-Meier plotter database analysis revealed that high expression of POP7/RPP20 was negatively associated with overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival (DMFS) in patients with breast cancer. Moreover, cellular functional experiments further demonstrated that knockout of endogenous POP7/RPP20 in MCF-7 and T47D breast cancer cells significantly suppressed breast cancer cell proliferation and colony formation. However, the underlying mechanisms for POP7/RPP20 in promoting breast cancer development and progression remain to be investigated. In the present project, we aimed to address the biological functions and related mechanisms for POP7/RPP20 in breast cancer development and progression using a variety of experimental models and techniques. To this end, the key scientific questions to be answered in this project include definition of biological functions of POP7/RPP20 in the process of breast cancer development and progression and elucidation of the effects of POP7/RPP20 on ribosome-protein synthesis and post-transcriptional regulation of cancer-related genes. The expected findings from this study will reveal the function as well as the molecular mechanism of POP7/RPP20 in breast cancer development and progression, and will provide a basis for discovering new strategies for inhibiting breast cancer development and progression. Taken together, in the light of the solid research background and preliminary data, we strongly believe that this study is definitely feasible, novel, and innovative and has the potential clinical translation implications.

阐明乳腺癌发生发展的分子机制以及鉴定治疗新靶点是目前乳腺癌研究领域亟需解决的关键科学问题之一。POP7/RPP20是核糖核酸酶P/MRP复合物蛋白亚单位之一，其在肿瘤发生发展中的作用及分子机制目前尚无相关报道。我们前期利用乳腺癌进展细胞模型结合RNA测序、CRISPR/Cas9敲除功能筛选、Kaplan-Meier plotter数据库生存分析以及细胞功能实验证实POP7/RPP20是一个潜在的乳腺癌促癌蛋白，但其分子机制有待进一步深入研究。本项目拟在此基础上系统性地解析POP7/RPP20在乳腺癌发生发展的功能及其分子机制。拟解决的关键科学问题包括明确POP7/RPP20在体内外对乳腺癌发生发展的影响、阐明其对核糖体-蛋白质合成以及基因转录后调控的影响及分子机理。本项目的研究成果将为揭示POP7/RPP20在乳腺癌发生发展中的功能及作用机制以及促进发现新的乳腺癌治疗靶点提供重要线索。

乳腺癌仍然是女性最为常见的恶性肿瘤，严重威胁着女性身心健康。RNA 结合蛋白(RBP)种类繁多、功能复杂，在转录后基因调控和表达中发挥着重要作用，其异常突变或异常结合产物与包括乳腺癌在内的诸多人类疾病相关。作为一种RNA结合蛋白，POP7是核糖核酸酶P/MRP的蛋白亚单位之一，目前对它的功能了解甚少，特别在肿瘤（包括乳腺癌）领域未见相关报道。因此，本课题拟研究POP7在乳腺癌中的具体功能和相关作用机制。本课题中，我们首先检测了RNA结合蛋白POP7在乳腺癌中的表达情况及其对临床预后的影响，我们发现POP7在乳腺癌细胞系中的表达量高于正常乳腺上皮细胞，其在乳腺癌患者组织中的表达量同样上调，并且可能与乳腺癌不良预后相关。接下来，通过一系列细胞功能实验，证实POP7的表达可以促进乳腺肿瘤细胞的增殖和克隆形成，也可以促进肿瘤细胞的迁移、侵袭能力；体内动物实验模型同样证实POP7可以促进乳腺癌的体内生长和转移。为了探索POP7的作用机制，我们通过RIP结合高通量测序技术（RIP-Seq）寻找POP7的靶标及其分布特征，结果表明POP7主要结合的靶标位于mRNA的内含子区域，另外，peak相关的基因主要影响了肿瘤相关的信号转导通路。进一步我们发现，POP7可以不同程度地影响其靶基因的mRNA稳定性。其中，POP7可以显著影响ILF3的mRNA稳定性及其蛋白表达水平，通过siRNA敲低ILF3表达进行细胞功能回复实验，表明POP7可以通过调节ILF3来发挥促进乳腺癌发展的作用。本课题首次研究了POP7在乳腺癌中的表达情况及其发挥促进肿瘤发展的功能，同时应用RIP-seq技术，以无偏差的方法系统地鉴定了POP7的结合靶标RNA分布和结合特征，并验证了ILF3作为POP7的靶标，其mRNA稳定性及表达水平受到POP7的影响，POP7通过调节ILF3而促进乳腺癌进展。因此，本课题对RNA结合蛋白POP7的功能和机制研究有望为乳腺癌新的治疗靶点提供线索。