Non-coding RNAs as Prognostic and Diagnostic Markers in Prostate Cancer

非编码RNA包括几种具有不同功能的RNA，包括微RNA和由超保守基因组区域（ucRNA）编码的RNA。MicroRNA表达在实体人肿瘤中通常发生改变。多种microrna已被证明具有致癌特性，或像肿瘤抑制基因一样起作用。这些microrna被称为oncomir。它们表达的改变与癌症的发展有因果关系，可以预测疾病的结局。最近，俄亥俄州立大学的Croce小组报告说，ucRNA的表达在人类癌症中也发生了改变。ucRNAs由481个超保守区域编码，这些区域被定义为大于200 bp的片段，在人类、大鼠和小鼠基因组中具有完美的保守性。与Croce博士合作，我们正在研究前列腺肿瘤的microRNA和ucRNA表达谱。我们也在研究选择的microrna的功能，例如miR-1， miR-106b-25簇和miR-200c-141簇，它们的表达在人类前列腺肿瘤中被发现改变，使用细胞培养和小鼠模型。最初，我们确定了60例原发性前列腺肿瘤和16例非肿瘤前列腺组织中microRNAs和mrna的全基因组表达。mRNA分析显示，microRNA加工的关键组分和几种microRNA宿主基因，如MCM7，在前列腺肿瘤中显著上调。与这些发现一致，肿瘤表达miR-106b-25簇，其映射到MCM7的内含子13，其水平显著高于非肿瘤前列腺。其他microrna的表达水平，包括miR-1-133簇、miR-200c和一些miR-106b-25簇同源物，在前列腺肿瘤中也发生了改变。在器官受限的肿瘤和前列腺外疾病扩展的肿瘤之间发现了microRNA丰度的其他差异，例如，在前列腺外扩展的肿瘤中miR-200功能同源物上调。最后，我们发现有证据表明，一些microrna是雄激素反应性的，肿瘤microrna影响癌前列腺中蛋白编码靶基因的转录丰度。总之，microRNA的表达随着前列腺癌的发生和进展而改变。其中一些microrna调节前列腺癌细胞中癌症相关基因的表达，并可能改变这些细胞的表型。在一项正在进行的研究中，我们正在分析这些样本中ucRNA的表达谱。初步结果表明，许多ucRNAs在肿瘤和周围正常组织、Gleason分级、器官局限性肿瘤和前列腺外病变之间存在差异表达。我们已经尝试用其他方法验证芯片数据，但迄今为止成功有限。这些问题可以解释为肿瘤与正常之间的ucRNAs表达差异较小，组织中许多ucRNAs的整体表达水平较低，以及ucRNA位点倾向于向正义和反义方向转录rna，这可能会干扰基于rt的定量分析的准确性。除了我们的ucRNA验证工作外，我们还确定了LNCaP前列腺癌细胞的ucRNA表达谱，这些细胞分别未经治疗或使用合成雄激素（R1881）、DNA低甲基化剂5-氮胞苷或组蛋白去乙酰化酶抑制剂曲古斯汀a。这些实验表明，一些ucRNA位点受到雄激素、DNA甲基化和染色质沉默的调节。我们继续研究miR-1、miR-106b-25和miR-200c-141在前列腺癌中的功能。miR-106b-25和miR-200c-141簇将在转基因小鼠中进行评估，这些簇在小鼠前列腺中通过probasin驱动表达。靶向载体已经构建完成，并正在细胞培养中进行评估，然后才能用于产生转基因小鼠。最终，我们将把miR-106b-25和miR-200c-141转基因小鼠导入易发生前列腺肿瘤的PTEN KO小鼠。本实验的目的是研究这两种microRNA簇如何调节PTEN KO小鼠前列腺癌的发展。在细胞培养中使用表型分析评估两个microrna。Mir-1在人类前列腺癌细胞系中过度表达，而内源性Mir-1的表达水平无法检测到。这些细胞中miR-1的上调导致生长停滞，并改变细胞对DNA损伤剂的敏感性。miR-1引起这些表型的机制目前正在研究中。在细胞培养模型中也研究了miR-106b的功能。我们之前观察到miR-106b-25簇抑制抗癌药物诱导的LNCaP细胞死亡。我们认为miR-106b是miR-106b-25簇抗凋亡作用的中介，并设计了实验来鉴定miR-106b抑制的凋亡基因。最近发表的一篇文章（Proc Natl Acad Sci USA 105:10513- 18,2008）发现，循环中的microrna是稳定的血液标志物，可能被证明对癌症检测有用。我们已经开始评估分析病例和对照组血浆样本中microRNA丰度的技术，以检查血液样本中的microRNA表达谱是否可以预测癌症的诊断和预后。在一项初步研究中，我们检测了来自癌症患者和人群对照的血浆样本中几种候选microrna的相对丰度。这些microRNA是从我们鉴定的肿瘤microRNA表达谱中选择的（mir-32, -106b, -141, -182, -196a, -199a, -200a, -200c）。初步结果表明，使用我们目前的总RNA分离和qRT-PCR检测的预扩增方案，大多数microrna都处于检测极限。未来的工作旨在改进我们的方案，例如，从我们的样品中去除肝素，并添加EGTA。与同事讨论发现肝素、Ca和Mg可能抑制microRNA测定。

Non-coding RNAs comprise several classes of RNA with diverse functions and include microRNAs and RNAs encoded by ultraconserved genomic regions (ucRNA). MicroRNA expression is commonly altered in solid human tumors. Multiple microRNAs have been shown to have oncogenic properties, or act like tumor suppressor genes. These microRNAs have been termed oncomiRs. An alteration in their expression is causatively linked to cancer development and can predict disease outcome. Recently, the Croce group at Ohio State University reported that expression of ucRNA is also altered in human cancer. ucRNAs are encoded by 481 ultraconserved regions that are defined as segments greater than 200 bp with perfect conservation among human, rat, and mouse genomes. In collaboration with Dr. Croce, we are investigating microRNA and ucRNA expression profiles of prostate tumors. We are also studying the function of selected microRNAs, e.g., miR-1, miR-106b-25 cluster, and the miR-200c-141 cluster, whose expression was found to be altered in human prostate tumors, using cell culture and mouse models. Initially, we had determined genome-wide expression of microRNAs and mRNAs in 60 primary prostate tumors and 16 non-tumor prostate tissues. The mRNA analysis revealed that key components of microRNA processing and several microRNA host genes, e.g., MCM7, were significantly up-regulated in prostate tumors. Consistent with these findings, tumors expressed the miR-106b-25 cluster, which maps to intron 13 of MCM7, at significantly higher levels than non-tumor prostate. The expression levels of other microRNAs, including the miR-1-133 cluster, miR-200c, and a number of miR-106b-25 cluster homologues, were also altered in prostate tumors. Additional differences in microRNA abundance were found between organ-confined tumors and those with extraprostatic disease extension, e.g., up-regulation of miR-200 functional homologues in tumors with extraprostatic extension. Lastly, we found evidence that some microRNAs are androgen-responsive and that tumor microRNAs influence transcript abundance of protein-coding target genes in the cancerous prostate. In summary, microRNA expression becomes altered with the development and progression of prostate cancer. Some of these microRNAs regulate the expression of cancer-related genes in prostate cancer cells and may alter the phenotype of these cells. In an ongoing study, we are analyzing the expression profiles for ucRNA in these samples. Preliminary results indicate that numerous ucRNAs are differentially expressed between tumor and surrounding normal tissue, by Gleason grade, and between organ-confined tumors and those with extraprostatic disease extension. We have attempted to validate the chip data by other methods, but so far had limited success. The problems are explained by the small differential expression of ucRNAs between tumor and normal, the overall low expression of many ucRNAs in the tissues, and the tendency of ucRNA loci to transcribe RNAs in sense and antisense direction, which can interfere with the accuracy of RT-based quantitative assays. In addition to our ucRNA validation work, we determined the ucRNA expression profile of LNCaP prostate cancer cells that were either untreated or treated with a synthetic androgen (R1881), the DNA hypomethylation agent, 5-azacytidine, or the histone deacetylase inhibitor, trichostatin A. These experiments revealed that several ucRNA loci are regulated by androgen, DNA methylation, and by chromatin silencing. We have continued our work to elucidate the function of miR-1 and the miR-106b-25 and miR-200c-141 clusters in prostate cancer. The miR-106b-25 and miR-200c-141 clusters will be evaluated in transgenic mice with a probasin-driven expression of these clusters in the mouse prostate. The targeting vectors have been constructed and are being evaluated in cell culture before they can be used to generate transgenic mice. Ultimately, we will cross the miR-106b-25 and miR-200c-141 transgenic mice into PTEN KO mice that are prone to develop prostate tumors. It is the aim if this experiment to examine how the two microRNA clusters modulate prostate cancer development in the PTEN KO mice. Two microRNAs are evaluated in cell culture using phenotypic assays. Mir-1 is being over-expressed in human prostate cancer cell lines that do not have any detectable levels of endogenous miR-1 expression. Up-regulation of miR-1 in these cells leads to growth arrest and alters the sensitivity of the cells to DNA damaging agents. The mechanism by which miR-1 causes these phenotypes is currently studied. The function of miR-106b is also studied in cell culture models. We previously observed that the miR-106b-25 cluster inhibits anticancer drug-induced cell death in LNCaP cells. We think that miR-106b is the mediator of the anti-apoptotic effects of the miR-106b-25 cluster and designed experiments to identify the apoptosis genes that are inhibited by miR-106b. A recent publication (Proc Natl Acad Sci USA 105:10513-18, 2008) found that circulating microRNAs are stable blood-based markers that may prove useful for cancer detection. We have begun evaluating the technology for analyzing microRNA abundance in plasma samples from cases and controls to examine whether the microRNA expression profile of blood samples is predictive for cancer diagnosis and prognosis. In a pilot study, we examined the relative abundance of several candidate microRNAs in plasma samples from cancer patients and population-based controls. These microRNAs were selected from the tumor microRNA expression profile that we identified (mir-32, -106b, -141, -182, -196a, -199a, -200a, -200c). Preliminary findings indicate that most microRNAs are at the detection limit using our current protocol of total RNA isolation and pre-amplification for the qRT-PCR assay. Future work is aimed to improve our protocol, e.g., removing heparin from our samples, and adding EGTA. Discussions with colleagues revealed that heparin, Ca, and Mg may inhibit the microRNA assays.