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

我们之前与俄亥俄州立大学的Carlo Croce博士合作，在60例原发性人类前列腺肿瘤和16例非肿瘤前列腺组织中检测了microRNAs和mrna的全基因组表达。分析显示，与周围非癌组织相比，前列腺肿瘤中microRNA加工的关键成分和许多microRNA都发生了显著改变。与非癌组织相比，肿瘤microRNA上调和下调，肿瘤的表达谱产生了诊断性microRNA特征。值得注意的是，前列腺肿瘤倾向于以显著高于非肿瘤前列腺的水平表达miR-106b-25集群的所有成员，这与miR-106b-25集群在前列腺肿瘤生物学中具有致癌特性是一致的。我们继续研究miR-106b-25集群的具体功能。对一个大型的公开可用的前列腺癌microRNA表达数据集的分析显示，肿瘤中miR-106b-25簇的表达增加与疾病进展相关。此外，肿瘤miR-106b表达升高与早期疾病复发相关，原发肿瘤中miR-106b高表达和CASP7 （caspase-7）低表达的结合是早期疾病复发的独立预测因子（校正风险比= 4.1；95%可信区间：1.6 - 12.3）。为了鉴定miR-106b的未知致癌功能，我们在LNCaP人类前列腺癌细胞中过表达miR-106b，以检测miR-106b诱导的蛋白编码基因的全局表达变化。该方法发现caspase-7是miR-106b的直接靶点，这一点通过Western blot分析和3'UTR报告基因试验得到证实。此外，当miR-106b和caspase-7的表达被抑制时，DU145人前列腺癌细胞中miR-106b敲低诱导的选择表型不发生。进一步的分析表明，在多个数据集中，caspase-7在原发性前列腺肿瘤和转移性病变中下调，并且caspase-7本身与疾病复发和疾病特异性生存有关。利用生物信息学，我们还观察到miR-106b-25可能特异性影响局灶黏附相关途径。使用mir -106b-25转导的22Rv1人前列腺癌细胞对这一观察结果进行了实验检验。用miR-106b-25慢病毒表达构建体感染后，22Rv1细胞对基底膜和骨基质相关细丝的粘附增强，软琼脂生长增强。总之，miR-106b-25被发现与前列腺癌的进展有关，并且可能通过改变细胞凋亡和局灶黏附相关的途径来实现。我们的研究结果表明，miR-106b-25簇在人类前列腺癌中起到致癌基因的作用，是早期复发的预测因子。在第二个项目中，我们使用微阵列表征了超保守区域衍生的非编码rna （ucRNAs）在前列腺肿瘤和周围正常组织中的表达谱。超保守区（UCR）是在哺乳动物物种中进化保守的超过200个碱基对的基因组片段。它们被认为具有转录增强子和选择性剪接调节器的功能。最近，研究表明许多rna都是从这些区域转录的。这些ucr编码转录本（ucRNAs）被发现以组织和疾病特异性的方式表达，并可能通过RNA:RNA相互作用干扰其他RNA的功能。我们假设ucRNAs在人类前列腺癌的发病机制中具有未知的作用。在一项初步研究中，我们检测了人类前列腺肿瘤中的ucRNA表达谱。我们使用一个带有962个代表481个人ucr的正义和反义序列的定制芯片，检测了ucRNA在切除的新鲜冷冻的人前列腺组织（57个肿瘤组织，7个非癌性前列腺组织）和用表观遗传药物（低甲基化剂5-Aza 2'脱氧胞苷和组蛋白去乙酰化酶抑制剂trichostatin a）或合成雄激素R1881处理的培养的前列腺癌细胞中的表达。选择的ucRNAs的表达也通过qRT-PCR和基于nanostring的检测进行评估。由于ucRNA可能通过直接和抑制RNA:RNA相互作用作为靶向蛋白质编码基因的RNA，因此应用计算分析来鉴定候选的ucRNA:mRNA结合对。我们观察到ucRNA在前列腺癌中的表达改变。106 +,加州大学。477 +,加州大学。363 +,加州大学。454+A)，在控制了错误发现（许多转录本的错误发现率为5%）后，发现这些ucRNAs与癌症发展、Gleason评分和前列腺外展有关。我们还发现了几种对表观遗传药物或雄激素治疗有反应的ucRNAs （R1881）。例如，LNCaP对人前列腺癌细胞的实验表明，uc。287+由R1881诱导；283+A在5-Aza 2'脱氧胞苷和trichostatin A联合治疗后表达上调。另外，计算分析预测了302种不同的义和反义ucRNAs与1058种不同mrna的RNA环环相互作用，推断了ucRNAs通过与mrna的直接相互作用可能具有的功能。我们从人类前列腺癌中ucRNA表达的首次研究中得出结论，该疾病具有这些rna的异常转录表达。目前，我们的重点是探索microRNAs如何影响前列腺癌细胞的代谢。该项目将初步确定与疾病复发相关的候选microrna。如果它们针对代谢途径中的基因，那么将对排名靠前的候选药物进行计算评估。预测会改变特定代谢途径的microRNA将通过过表达和敲除方法进行实验评估，以促进对microRNA表达变化的癌症转录组和代谢组的综合评估。

We had previously examined genome-wide expression of microRNAs and mRNAs in 60 primary human prostate tumors and 16 non-tumor prostate tissues in collaboration with Dr. Carlo Croce at Ohio State University. The analysis revealed that both key components of microRNA processing and numerous microRNAs were significantly altered in prostate tumors when compared with surrounding non-cancerous tissue. Tumor microRNAs were up- and down-regulated when compared with non-cancerous tissue and the expression profile of the tumors yielded a diagnostic microRNA signature. Notably, prostate tumors tended to express all members of the miR-106b-25 cluster at significantly higher levels than non-tumor prostate, which is consistent with the miR-106b-25 cluster having oncogenic properties in prostate tumor biology. We continued to study the specific functions of the miR-106b-25 cluster. Analyzing a large publically available microRNA expression dataset for prostate cancer revealed increased expression of the miR-106b-25 cluster in tumors that correlated with disease progression. Moreover, increased tumor miR-106b expression was associated with early disease recurrence and the combination of high miR-106b and low CASP7 (caspase-7) expression in primary tumors was an independent predictor of early disease recurrence (adjusted hazard ratio = 4.1; 95% confidence interval: 1.6 to 12.3). To identify yet unknown oncogenic functions of miR-106b, we overexpressed miR-106b in LNCaP human prostate cancer cells to examine miR-106b-induced global expression changes among protein-coding genes. The approach revealed that caspase-7 is a direct target of miR-106b, which was confirmed by Western blot analysis and a 3'UTR reporter assay. Moreover, selected phenotypes induced by miR-106b knockdown in DU145 human prostate cancer cells did not develop when both miR-106b and caspase-7 expression were inhibited. Further analyses showed that caspase-7 is down-regulated in primary prostate tumors and metastatic lesions across multiple datasets and is by itself associated with disease recurrence and disease-specific survival. Using bioinformatics, we also observed that miR-106b-25 may specifically influence focal adhesion-related pathways. This observation was experimentally examined using miR-106b-25-transduced 22Rv1 human prostate cancer cells. After infection with a miR-106b-25 lentiviral expression construct, 22Rv1 cells showed increased adhesion to basement membrane- and bone matrix-related filaments and enhanced soft agar growth. In summary, miR-106b-25 was found to be associated with prostate cancer progression and may do so by altering apoptosis- and focal adhesion-related pathways. Our findings indicate that the miR-106b-25 cluster acts like an oncogene in human prostate cancer and is a predictor of early recurrence. In a second project, we characterized the expression profile of ultraconserved region-derived non-coding RNAs (ucRNAs) in prostate tumors and surrounding normal tissue using microarrays. Ultraconserved regions (UCR) are genomic segments of more than 200 base pairs that are evolutionarily conserved among mammalian species. They are thought to have functions as transcriptional enhancers and regulators of alternative splicing. Recently, it was shown that numerous RNAs are transcribed from these regions. These UCR-encoded transcripts (ucRNAs) were found to be expressed in a tissue- and disease-specific manner and may interfere with the function of other RNAs through RNA:RNA interactions. We hypothesized that ucRNAs have unidentified roles in the pathogenesis of human prostate cancer. In a pilot study, we examined ucRNA expression profiles in human prostate tumors. Using a custom microarray with 962 probesets representing sense and antisense sequences for the 481 human UCRs, we examined ucRNA expression in resected, fresh-frozen human prostate tissues (57 tumors, 7 non-cancerous prostate tissues) and in cultured prostate cancer cells treated with either epigenetic drugs (the hypomethylating agent, 5-Aza 2'deoxycytidine, and the histone deacetylase inhibitor, trichostatin A) or a synthetic androgen, R1881. Expression of selected ucRNAs was also assessed by qRT-PCR and NanoString-based assays. Because ucRNAs may function as RNAs that target protein-coding genes through direct and inhibitory RNA:RNA interactions, computational analyses were applied to identify candidate ucRNA:mRNA binding pairs.We observed altered ucRNA expression in prostate cancer (e.g., uc.106+, uc.477+, uc.363+A, uc.454+A) and found that these ucRNAs were associated with cancer development, Gleason score, and extraprostatic extension after controlling for false discovery (false discovery rate 5% for many of the transcripts). We also identified several ucRNAs that were responsive to treatment with either epigenetic drugs or androgen (R1881). For example, experiments with LNCaP human prostate cancer cells showed that uc.287+ is induced by R1881 whereas uc.283+A was up-regulated following treatment with combined 5-Aza 2'deoxycytidine and trichostatin A. Additional computational analyses predicted RNA loop-loop interactions of 302 different sense and antisense ucRNAs with 1058 different mRNAs, inferring possible functions of ucRNAs via direct interactions with mRNAs. We conclude from this first study of ucRNA expression in human prostate cancer that the disease has aberrant transcript expression of these RNAs. Currently, our focus is exploring how microRNAs may affect the metabolism in prostate cancer cells. This project will initially identify candidate microRNAs that are associated with disease recurrence. Top-ranked candidates will then be assessed computationally if they target genes in metabolic pathways. microRNAs that are predicted to alter specific metabolic pathways will then be experimentally evaluated using overexpression and knockout approaches to facilitate an integrated assessment of the cancer transcriptome and metabolome in response to microRNA expression changes.