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

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.

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。

本研究首先系统性综述小激活RNA如何在细胞内上调基因表达的分子机制，及RNA激活作为分子工具在临床治疗各类疾病的应用前景。研究第二部分探索新的临床有意义的小分子非编码RNA, miR-1-3p参与前列腺癌细胞增殖及进展的分子机制。研究发现miR-1-3p在前列腺癌组织及细胞内低表达，并且与前列腺癌患者无疾病进展生存呈明显负相关；过表达miR-1-3p可以抑制前列腺 癌22RV1和 LncaP细胞系及前列腺癌细胞体外成瘤的增殖；机制研究表明miR-1-3p 直接靶向细胞周期相关蛋白E2F5和PFTK1的3’-UTR 并抑制两种蛋白的表达；敲低E2F5和PFTK1抑制前列腺癌细胞增殖，而同时敲低miR-1-3p和上述两蛋白表达则部分逆转了上述效应，从而明确了miR-1-3p通过靶向E2F5和PFTK1来调控前列腺癌细胞增殖的分子机制。研究最后一部分，我们观察HnRNPK蛋白在前列腺癌中高表达且与患者PSA的水平及复发率相关，多个人源前列腺癌细胞系及原发性前列腺癌患者组织样本中进一步证实HnRNP K mRNA及蛋白的高表达；前列腺癌细胞系稳定敲低HnRNPK的表达明显抑制细胞增殖和细胞周期；miR-206 和miR-613在前列腺癌细胞系表达下调且直接靶向HnRNPK 3’-UTR, 过表达上述miRNA可抑制前列腺癌细胞增殖；进而明确Cullin 3 SPOP E3 泛素连接酶负性调控HnRNPK 蛋白稳定性；且前列腺癌患者相关的SPOP突变不能泛素化降解HnRNPK蛋白。上述研究首先揭示小RNA分子参与前列腺癌发生发展的分子机制，其次揭示HnRNPK蛋白在前列腺癌异常蓄积及其转录后水平及翻译后水平调控的分子机制，为HnRNPK蛋白作为可能的靶点研发，用于临床治疗SPOP突变的前列腺癌患者提供分子理论依据。