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

非编码 RNA 作为前列腺癌的预后和诊断标志物

• 批准号: 8937884
• 负责人: Stefan Ambs
• 金额: $ 7.44万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937884
• 关键词: 3' Untranslated Regions; Adhesions; Agar; Alternative Splicing; Androgen Analogues; Androgens; Apoptosis; Base Pairing; Basement membrane; Binding; Bioinformatics; Biological Assay; Bone Matrix; Cell Culture Techniques; Cells; Code; Collaborations; Confidence Intervals; Custom; DU145; Data Set; Deoxycytidine; Development; Diagnostic; Disease; Disease Progression; Enhancers; Epigenetic Process; Extraprostatic; Filament; Focal Adhesions; Freezing; Functional RNA; Genes; Genetic Transcription; Genomic Segment; Gleason Grade for Prostate Cancer; Goals; Growth; Histone Deacetylase Inhibitor; Human; Infection; LNCaP; Malignant Neoplasms; Malignant neoplasm of prostate; Messenger RNA; Metastatic Lesion; MicroRNAs; Molecular Profiling; Normal tissue morphology; Ohio; Oncogenes; Oncogenic; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Pilot Projects; Primary Neoplasm; Process; Prognostic Marker; Property; Prostate; Prostatic Neoplasms; Proteins; RNA; Recurrence; Reporter; Resected; Role; Ru-1881; Tissues; Transcript; Trichostatin A; Tumor Biology; Universities; Western Blotting; Xenograft Model; base; caspase-7; genome-wide; hazard; member; nano-string; overexpression; prognostic; prostate cancer cell; protein expression; research study; tumor; tumor progression

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.

我们以前曾与俄亥俄州立大学的Carlo Croce博士合作研究了60种原发性人类前列腺肿瘤和16个非肿瘤前列腺组织中MicroRNA和mRNA的全基因组表达。分析表明，与周围的非癌组织相比，前列腺肿瘤的microRNA加工和许多microRNA的关键成分都显着改变。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明显高于非肿瘤前列腺水平来表达miR-106b-25簇的所有成员，这与前列腺肿瘤生物学中具有致癌特性的miR-106B-25簇一致。我们继续研究miR-106b-25簇的特定功能。分析用于前列腺癌的大型公共可用的microRNA表达数据集发现，在与疾病进展相关的肿瘤中，miR-106b-25簇的表达增加。此外，肿瘤miR-106b表达增加与早期疾病复发有关，而在原发性肿瘤中高miR-106b和低CASP7（Caspase-7）表达的组合是早期疾病复发的独立预测指标（调整后危险比= 4.1; 95％置信区间； 95％置信区间：1.6至12.3）。为了鉴定miR-106b的尚未知道的致癌功能，我们在LNCAP人前列腺癌细胞中过表达miR-106b，以检查miR-106b诱导的蛋白质编码基因中的全球表达变化。该方法表明，caspase-7是miR-106b的直接靶标，它通过Western blot分析和3'UTR报告基因测定得到了证实。此外，当miR-106b和caspase-7表达均被抑制时，DU145的miR-106b敲低诱导的选定表型不会发展。进一步的分析表明，caspase-7在多个数据集的原发性前列腺肿瘤和转移性病变中被下调，并且与疾病复发和疾病特异性生存率本身就是相关的。使用生物信息学，我们还观察到miR-106b-25可能会特别影响与局灶性粘附相关的途径。使用miR-106b-25转导的22RV1人前列腺癌细胞对该观察结果进行了实验检查。在用miR-106b-25慢病毒表达构建体感染后，22RV1细胞显示出对基底膜和骨基质相关的丝的粘附增加，并增强了软琼脂的生长。总而言之，发现miR-106b-25与前列腺癌的进展有关，并且可以通过改变与局灶性粘附相关的途径来做到这一点。我们的发现表明，miR-106b-25簇的作用像是人类前列腺癌中的癌基因，并且是早期复发的预测指标。在第二个项目中，我们使用微阵列在前列腺肿瘤和周围正常组织中表征了超保存区域衍生的非编码RNA（UCRNA）的表达曲线。超保守区域（UCR）是哺乳动物物种中进化保守的200多个碱基对的基因组段。人们认为它们具有替代剪接的转录增强子和调节剂的功能。最近，显示出许多RNA是从这些区域转录的。发现这些UCR编码的转录本（UCRNA）以组织和疾病特异性的方式表达，并可能通过RNA：RNA相互作用来干扰其他RNA的功能。我们假设UCRNA在人类前列腺癌的发病机理中具有未识别的作用。在一项试点研究中，我们检查了人类前列腺肿瘤中的UCRNA表达谱。我们使用具有962个探针的定制微阵列，代表481人UCR的含义和反义序列，我们检查了在切除的，新鲜的肉体前列腺组织（57种肿瘤，7种非癌性前列腺组织）中的UCRNA表达，并在用培养的前列腺癌和用催眠的药物（催眠型和5--催眠型）中治疗的培养的前列腺癌中，并组蛋白脱乙酰基酶抑制剂trichostatin A）或合成雄激素R1881。还通过QRT-PCR和基于纳米弦的测定法评估了所选UCRNA的表达。由于UCRNA可能充当RNA，通过直接和抑制性RNA靶向蛋白质编码基因：RNA相互作用，因此应用计算分析来识别候选UCRNA：mRNA结合对。我们观察到UCRNA在前列腺癌中的表达改变了，例如UC.106+，UC.477+，UC.477+，UC.363+a，UC.444444444444444444444444444444444444444.4444444444444444444444年4月44日控制错误发现后，与癌症的发展，格里森评分和外压外扩展相关（许多成绩单的错误发现率为5％）。我们还确定了几种对用表观遗传药物或雄激素治疗有反应的UCRNA（R1881）。例如，通过LNCAP人前列腺癌细胞进行的实验表明，UC.287+是由R1881诱导的，而UC.283+ A在用5-Aza 2'ddeoxytidine和trichostatin合并后进行了上调。通过与mRNA直接相互作用，UCRNA的可能功能。我们从人类前列腺癌中UCRNA表达的第一项研究中得出结论，该疾病的转录本表达异常。