HnRNP A2/B1在乳腺癌细胞中的作用及分子机制

HnRNP A2/B1 participates in virtually all aspects of mRNA processing, including alternative splicing，nucleocytoplasmic trafficking and translational regulation of specific mRNAs. In our preliminary works, it has been found that hnRNP A2/B1 was a common nuclear matrix protein differentially expressed in many tumor cells during induced differentiation. And the nucleocytoplasmic shuttling of hnRNP A2/B1 and its co-localization with several proteins related to the differentiation were found in tumor cells such as breast cancer. These results suggest that hnRNP A2/B1 was a key regulatory protein during canceration and reversion regulation. But the specific mechanism under which it works remains to be answered. Based on the works done before, the present project aims at carrying out further studies in the following aspects: ① To reveal the principles of hnRNP A2/B1 isoforms expression in tumor cells; ② To carry out proteomics study of hnRNP A2/B1 interacting proteins so as to build a network of hnRNP A2/B1 interactions; ③ To investigate the types and the alternative splicing of the mRNAs, which are the downstream targets of hnRNPA2/B1, and to analyze the pathways affected by mRNAs; ④ To illustrate the effect of hnRNP A2/B1 suppression on the malignant phenotype of breast cancer. The above-mentioned researches aim at revealing the expression of hnRNP A2/B1 isoforms, its interacting proteins network and downstream target genes in tumor cells so as to clarify its regulating mechanism in tumor cells. These studies will help to reveal hnRNPA2/B1's mechanism in breast cancer and provide a new target for researches on the mechanism, clinical diagnosis, prevention and treatment of tumor.

HnRNP A2/B1参与特定mRNA的可变剪接、核质转运与翻译调控。我们前期工作发现：hnRNP A2/B1是多种癌细胞诱导分化后出现的共同差异核基质蛋白；在乳腺癌等肿瘤中发生核-质定位变化并与多种分化相关蛋白共定位。前期结果提示它是细胞癌变与逆转的一个关键调控节点，但具体机制不清楚。本课题拟研究:① 肿瘤细胞中hnRNP A2/B1的异构蛋白表达规律；②hnRNPA2/B1异构蛋白的相互作用蛋白质组，构建相互作用蛋白分子网络；③ hnRNPA2/B1异构蛋白调控的下游靶mRNA种类及剪接型，分析其参与调控的通路网络；④ hnRNP A2/B1沉默对乳腺癌细胞恶性表型的影响。通过上述研究，揭示肿瘤细胞中hnRNP A2/B1异构蛋白表达规律、相互作用蛋白网络及下游靶mRNA涉及的通路网络，从而阐明hnRNPA2/B1在乳腺癌中作用机制，为肿瘤的机理研究、临床诊断和治疗提供一个新靶点。

hnRNPA2/B1参与RNA加工、可变剪接和基因调控，在多种肿瘤中高表达，但其乳腺癌中作用与机制还不清楚。本项目为了探究其在乳腺癌中的具体作用与机制，通过CRISPR/CAS9构建稳定敲除hnRNPA2/B1的MCF-7和MDA-MB-231细胞系，检测其对增殖、迁移和侵袭等重要功能表型的影响，并进行了mRNA、非编码RNA和环状RNA的测序分析研究，和hnRNPA2/B1互作蛋白的组学研究，分析研究其影响的分子机制；在体内的动物研究模型上，我们通过裸鼠移植瘤实验，研究了A2/B1对乳腺癌细胞的成瘤能力，生长能力的影响。.细胞模型研究结果：增殖与凋亡实验、transwell等实验结果显示，敲除hnRNPA2/B1会抑制细胞的增殖和凋亡，但却促进细胞的迁移和侵袭能力。这些变化的表型可以被重新表达的hnRNPA2/B1逆转。 Western blot和定量PCR对相关分子标记基因的检测也证实了上述结论。动物实验结果：敲除hnRNPA2/B1显著影响了乳腺癌细胞的成瘤能力和血管生成能力。组学研究结果： hnRNPB1相互作用蛋白进行KEGG信号通路及功能分析，hnRNPB1相互作用蛋白主要涉及到的KEGG信号通路有RNA运输、RNA的降解、TCA循环、细胞增殖、氧化磷酸化、丙酮酸代谢、阿尔滋海默氏病、蛋白酶体、细胞周期的进程、细胞粘附、细胞凋亡等。转录组测序结果提示，敲除hnRNPA2/B1抑制了多个增殖、凋亡、迁移和侵袭有关的信号通路。可变剪接分析显示，与迁移和凋亡相关的RON、Caspase9、c-FLIP、PLK3和Bim等基因的mRNA的可变剪接受到了hnRNPA2/B1的影响，可能参与了迁移和凋亡的调控。.本项目研究结果显示，hnRNPA2/B1是乳腺癌的关键调控蛋白，它的高表达促进癌细胞的增殖，低表达却能促进肿瘤的转移，这可能是通过它影响的基因可变间接事件实现的。hnRNPA2/B1在乳腺癌中的独特作用可能参与了乳腺癌异质性的形成，可望成为乳腺癌治疗的重要靶点。