hnRNP家族蛋白及相关蛋白质参与小RNA激活前列腺癌细胞株p21基因表达的机制研究

Clarification of the mechanisms underlying RNA activation (RNAa) is important for the selection of small-activating RNA (saRNA) for a particular target gene and its application in clinical settings such as for the treatment of cancer and metabolic diseases. However, exactly how RNAa upregulates target gene expression largely remains elusive. In the previous study, we demonstrated that dsP21-322, a small activated RNA, activates p21 gene expression by targeted binding to its promoter sequence. We found that five specific proteins are implicated in this process. Among which, the heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2/B1) protein was found to play an essential role in dsP21-322-mediated p21 expression, while the functions for other four proteins are yet to be addressed. The goal of this proposal is to employ the human prostate adenocarcinoma cell line as a model along with the application of co-immunoprecipitation(Co-IP), Pull-down,RNA interference (RNAi)and chromatin immunoprecipitation (ChIP), to fully dissect the role of these four proteins in dsP21-322-mediated p21 transcription and identify the association of these prpteins with the dsP21-322 target site on p21 promoter.These studies would be important for better understanding of the detailed mechanisms underlying RNAa. They also provide important theoretical basis and effective means for RNAa application.

阐明RNA激活（RNAa）机制不仅有利于选择调控靶基因表达的RNAa,而且对其临床应用如肿瘤及代谢性疾病的治疗意义重大，但RNAa的确切机制至今尚未明确。我们前期研究发现小激活RNA,dsP21-322能特异地与p21基因启动子区DNA结合，进而激活p21的表达。而这一过程可能涉及5种特异性蛋白质包括核内不均一核糖核蛋白A2/B1(hnRNPA2/B1)、A1(hnRNPA1)、C1/C2(hnRNPC1/C2)、核磷蛋白1及组蛋白簇1等，并证实hnRNPA2/B1是RNAa发挥效应所必须蛋白。基于此，我们拟以前列腺癌细胞株为模型，采取免疫共沉淀，Pull-down,RNA干扰及染色质免疫共沉淀等技术，验证其余4种蛋白在RNAa过程中的作用及其与dsP21-322靶位点的联系。这些研究，可以进一步丰富和完善对RNAa机制的认识，为RNAa的应用提供重要的理论依据和有效手段。

近十年来的多个研究表明，RNA激活（RNAa）现象存在于秀丽隐杆线虫、鼠类及人类中，在进化过程中演化成一种保守的基因调控工具，能在转录水平参与基因表达调控，但其确切机制仍待明确。我们前期研究阐明了外源性小双链dsRNA（dsP21-322）在前列腺癌细胞系中能特异地富集p21基因启动子区，招募AGO2等蛋白至靶位点进而激活p21基因的表达，及hnRNP家族蛋白与dsP21-322相互联系，且hnRNPA2/B1参与其介导的RNAa过程等部分机制。但仍有部分机制有待进一步完善，如hnRNP家族蛋白在RNAa过程中的作用，dsRNA靶点及参与蛋白的深入验证及有无内源性MicroRNA上调p21的表达等。.本研究采用前列腺癌细胞株为模型，首先利用Co-IP和pull down明确hnRNPA1、hnRNPK等蛋白与dsP21-322之间的相互作用并不明显，提示两者可能并非结合dsRNA在RNAa过程中发挥作用。其次，采取荧光素酶报告实验、共聚焦成像、RNA干扰及染色质免疫共沉淀等技术，验证了dsRNA结合靶向启动子区在转录水平激活p21基因的表达，阐明靶位点区域有AGO1 和组蛋白修饰等相关蛋白的富集。最后，通过miRanda分析发现，miR-3619-5p和p21启动子区的碱基序列也有较高的互补性，证实了内源性的miR-3619-5p也能够通过靶向p21基因启动子区激活其表达并抑制前列腺癌细胞生长。上述研究结果，可以进一步丰富和完善对内外源性小RNA激活特定基因作用机制的认识，为更好的利用其特性上调基因表达治疗肿瘤等多种疾病提供重要的理论依据和有效手段。