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

在实体人类肿瘤中，microRNA的表达通常会发生改变。多个microRNAs已被证明具有致癌特性，或发挥类似肿瘤抑制基因的作用。这些microRNAs被称为oncomiRs。它们表达的变化与癌症的发展有因果关系，并可以预测疾病的结果。最近，Croce小组(俄亥俄州立大学医学博士卡洛·克罗齐)报告说，由超服务基因组区域(UcRNA)编码的RNA在人类癌症中的表达发生了变化。到目前为止，已经有481个这样的超冷冻元件被注释并定义为大于200bp的片段，在人类、大鼠和小鼠基因组中具有完美的保守性。与克罗齐博士合作，我们正在研究前列腺癌的microRNA和ucRNA表达谱。这一团队合作导致了第一项研究，即应用大规模基因表达谱来识别非裔美国人和欧洲裔美国人患者肿瘤中microRNA功能的变化。我们测定了60例原发性前列腺癌和16例非肿瘤前列腺癌组织中microRNAs和mRNAs的全基因组表达。MRNA分析表明，在前列腺癌中，microRNA加工的关键成分和几个microRNA宿主基因，如MCM7和C9orf5，显著上调。与这些发现一致的是，肿瘤表达miR-106b-25簇，它映射到MCM7的内含子13，以及miR-32，映射到C9orf5的内含子14，显著高于非肿瘤前列腺。其他microRNAs的表达水平，包括一些miR-106b-25簇同系物，在前列腺癌中也发生了变化。在器官受限的肿瘤和前列腺外疾病扩展的肿瘤之间，还发现了microRNA丰度的额外差异。最后，我们发现证据表明，一些microRNAs是雄激素反应的，并且肿瘤microRNAs影响前列腺癌中蛋白质编码靶基因的转录丰度。在细胞培养中，E2F1和p21/WAF1被鉴定为miR-106b、miR-32的BIM和miR-1的Exportin-6和蛋白酪氨酸激酶9的靶点。总之，随着前列腺癌的发展和进展，microRNA的表达也会发生变化。其中一些微小RNA调节前列腺癌细胞中癌症相关基因的表达。我们还比较了非裔美国人和欧洲裔美国人之间的肿瘤microRNA签名，但只有少数microRNA有差异表达。在非裔美国人的肿瘤中，miR-129、miR-196b和miR-342的含量低于欧洲裔美国人的肿瘤。从分析来看，肿瘤微RNA的表达似乎并没有因种族/民族的不同而有任何显著的差异。目前，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、器官受限的肿瘤和有前列腺外疾病扩展的肿瘤之间存在差异表达。芯片数据正在通过其他方法进行验证。最近的一份出版物(Proc Natl Acad Sci USA 105：10513-18,2008)发现，循环中的microRNAs大小仅为17-25bp，是稳定的血液标志物，可用于癌症检测。我们已经开始评估前列腺癌患者和对照的血清样本中microRNA丰度的分析技术，以检查血液样本的microRNA表达谱是否可以用于预测肿瘤的存在。在一项初步研究中，我们将评估癌症患者和人群对照血清样本中几种候选microRNAs的相对丰度。这些microRNA将从我们鉴定的肿瘤microRNA表达谱中挑选出来。这项研究的样本将来自我们正在进行的马里兰州大巴尔的摩前列腺癌病例对照研究。

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 (Carlo Croce, M.D., Ohio State University) reported that expression of RNAs encoded by ultraconserved genomic regions (ucRNA) is altered in human cancer. At this time, 481 of these ultraconserved elements have been annotated and 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. This teamwork led to the first study that applied large-scale gene expression profiling to identify alterations in microRNA function in tumors from African-American and European-American patients. We 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 and C9orf5, 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, and miR-32, which maps to intron 14 of C9orf5, at significantly higher levels than non-tumor prostate. The expression levels of other microRNAs, including 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. 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 cell culture, E2F1 and p21/WAF1 were identified as targets of miR-106b, Bim of miR-32, and exportin-6 and protein tyrosine kinase 9 of miR-1. 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. We also compared the tumor microRNA signatures between African-Americans and European-Americans, but only a few microRNAs were differentially expressed. miR-129, miR-196b, and miR-342 were found to be less abundant in tumors of African-Americans than in tumors of European-Americans. From the analysis, it did not appear that tumor microRNAs are differentially expressed to any major degree by race/ethnicity. Currently, 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. The chip data are in the process of being validated by other methods. A recent publication (Proc Natl Acad Sci USA 105:10513-18, 2008) found that circulating microRNAs, which are only 17-25 bp in size, are stable blood-based markers that could be useful for cancer detection. We have begun evaluating the technology for analyzing microRNA abundance in serum samples from prostate cancer cases and controls to examine whether the microRNA expression profile of blood samples can be used to predict the presence of tumor. In a pilot study, we will evaluate the relative abundance of several candidate microRNAs in serum samples from cancer patients and population-based controls. These microRNAs will be selected from the tumor microRNA expression profile that we identified. Samples for the study will be drawn from our ongoing case-control of prostate cancer in the greater Baltimore, Maryland area.