非编码RNA激活前列腺癌细胞株抑癌基因p21的转录组学和蛋白组学研究

非编码RNA(ncRNA)可以序列特异性地激活沉默或低表达基因，在基因功能研究及肿瘤基因靶向治疗方面极具潜力。我们一直深入研究ncRNA的激活机制，发现小激活RNA(saRNA)包括内外源性小RNA分子均可与其序列互补配对的启动子区DNA相联系，招募AGO蛋白至启动子区形成saRNA-AGO转录复合体，激活过程伴随表观遗传学改变。但ncRNA激活机制的核心问题如saRNA作用靶点及协同saRNA-AGO转录复合体调控的转录组学和蛋白质组学仍不明确。本研究以人前列腺癌细胞系PC-3和DU-145为研究对象，基因编辑抑癌基因p21启动子区明确saRNA靶向位点，ChIP、RIP结合高通量测序、串联质谱探明转录水平参与激活机制的lncRNA和蛋白组，利用RIP、RNA干扰揭示lncRNA与表观遗传学的内在机制。建立完善的ncRNA激活网络调控机制模型，为其靶向治疗肿瘤性疾病提供可行理论依据。

Non coding RNA (ncRNA) can specifically activate silenced or low expression genes, and thus has great potential in gene function research and tumor gene targeted therapy. We have studied ncRNA activation mechanism for many years and found that small activating RNA (saRNA) including the exogenous and endogenous small RNA molecules can both interact with its complementary promoter DNA sequence. This process is associated with RNA polymerase II and AGO protein recruitment to the saRNA-target site and formed saRNA-AGO transcription complex. Additionally, we found that the activating process is associated with epigenetics change at promoter region. However, the key issues of ncRNA activation mechanisms, such as the target for saRNA and the mechanisms of transcriptomic and proteomic regulation for the saRNA-AGO transcription complex, remain unclear. In this study, human prostate cancer cell lines PC-3 and DU-145 as our research object. CRISPR-Cas9 is used to edit tumor suppressor gene p21 promoter in order to clarify specifically targeting loci for saRNA. Moreover, we attempt to find the lncRNA and proteomic involved in ncRNA mechanisms through ChIP and RIP combined with high-throughput sequencing and mass tandem mass spectrometry methods. In addition, revealing the internal mechanism for epigenetics and lncRNA in ncRNA activation mechanism by using RIP and RNAi methods. These study will establish more perfect model of network regulation mechanism for ncRNA activation, and will also provide more important new theoretical basis for treating tumor diseases with activating ncRNA.