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: 8552878
• 负责人: Stefan Ambs
• 金额: $ 16.72万
• 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/8552878
• 关键词: Actins; Adhesions; Affect; Agar; Androgen Analogues; Apoptosis; Basement membrane; Bioinformatics; Biological Assay; Bone Matrix; Cell Culture Techniques; Cell Cycle Progression; Cell Proliferation; Cells; Code; Collaborations; Confidence Intervals; DNA biosynthesis; DU145; Data Set; Development; Diagnostic; Disease; Distant Metastasis; Down-Regulation; Epigenetic Process; F-Actin; Filament; Focal Adhesions; Functional RNA; Genes; Genetic Transcription; Goals; Growth; Histone Deacetylase Inhibitor; Human; Hypermethylation; In Vitro; Infection; LNCaP; Lead; Malignant Neoplasms; Malignant neoplasm of prostate; Measures; Mediating; Messenger RNA; Metastatic Lesion; Methods; MicroRNAs; Mitosis; Molecular Profiling; Mus; Normal tissue morphology; Ohio; Oncogenes; Oncogenic; Pathway interactions; Patients; Pattern; Phenotype; Primary Neoplasm; Process; Prognostic Marker; Property; Prostate; Prostatic Neoplasms; Proteins; RNA Interference; Recurrence; Recurrent disease; Reporter; Role; Small Interfering RNA; Systems Analysis; Technology; Time; Tissues; Transcript; Tumor Biology; Tumor Suppressor Proteins; Universities; Untranslated Regions; Western Blotting; Xenograft Model; base; cancer cell; caspase-7; cell motility; follow-up; gene repression; genome-wide; hazard; mRNA Expression; member; mouse model; nano-string; novel; overexpression; prognostic; promoter; protein expression; repaired; 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. The expression of miR-1 and miR-133 was consistently lower in tumors than in non-tumor prostate, indicating that these microRNAs may act as tumor suppressors. In a follow-up study for miR-1-133 cluster expression in human prostate tumors, we further corroborated this finding in an independent dataset and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the candidate tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer cells and the analysis of tumors indicated promoter hypermethylation of the miR-1 locus in a subset of human tumors. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair, and actin dynamics. This observation was further corroborated with protein expression analysis and 3-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters gamma-H2A.X marker expression and affects the cellular organization of F-actin and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.We also continued to study the specific functions of the miR-106b-25 cluster. Analyzing a large microRNA expression dataset for prostate tumor from patients with recurrent disease status revealed increased expression of the miR-106b-25 cluster in these tumors. Moreover, the cluster was found to be up-regulated in distant metastases of the prostate and in prostate tumors of TRAMP mice. 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, which is recapitulated in the TRAMP mouse model, and is a predictor of early recurrence.We have previously characterized the expression profile of ultraconserved region-derived non-coding RNAs (ucRNAs) in prostate tumors and surrounding normal tissue using microarrays. We also found that some key ucRNAs are silenced by epigenetic mechanisms in human prostate cancer cells. Our lab is the first to explore genome-wide expression profiles of ucRNAs in prostate cancer and one of the first to explore regulatory mechanisms of these RNAs. However, quantification of ucRNAs by other methods than microarrays has been challenging. Recently, the use of the NanoString nCounter Analysis System allowed us the direct quantification of ucRNA transcripts with this newly introduced technology that may rival quantitative real-time PCR and array based assays because of its ability to measure RNAs in a direct and absolute manner. Other experiments showed that ucRNA transcription patterns can be modified by (synthetic) androgen exposure and siRNA-based down-regulation of candidate ucRNAs can lead to global mRNA expression changes, indicating that ucRNAs may have a broad functional role in prostate cancer.

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。在肿瘤中，miR-1和miR-133的表达始终低于非肿瘤前列腺，表明这些microRNA可能充当肿瘤抑制子。在人类前列腺肿瘤中miR-1-133簇表达的后续研究中，我们在独立数据集中进一步证实了这一发现，并使新的观察结果表明，远处转移中miR-1表达进一步降低，并且是疾病复发的预测指标。此外，我们进行了体外实验，以探索miR-1的候选肿瘤抑制功能。基于细胞的测定表明，在人类前列腺癌细胞中，miR-1表观遗传沉默，肿瘤的分析表明在人类肿瘤的一部分中，miR-1基因座的启动子高甲基化。这些细胞中miR-1的过表达导致在调节细胞周期进程，有丝分裂，DNA复制/修复和肌动蛋白动力学的途径中的基因生长抑制和下调。通过蛋白质表达分析和基于3-UTR的记者测定法进一步证实了这一观察结果，表明这些途径中的基因是miR-1的直接或间接靶标。基因集富集分析表明，miR-1介导的肿瘤抑制作用在全球范围内与组蛋白脱乙酰基酶抑制剂相似。最后，我们获得了初步证据，表明miR-1会改变伽马 - h2a.x标记表达并影响F-肌动蛋白和菲律宾形成的细胞组织。总之，我们的发现表明，miR-1通过影响多个与癌症相关的过程并抑制细胞增殖和运动性来充当前列腺癌中的肿瘤抑制因子。我们还继续研究miR-106B-25簇的特定功能。分析来自复发性疾病状态患者的前列腺肿瘤的大型microRNA表达数据集显示，这些肿瘤中miR-106B-25簇的表达增加。此外，发现该簇在前列腺的遥远转移和流浪小鼠的前列腺肿瘤中被上调。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，在流浪汉模型中被概括，并且是早期复发的预测因子。我们先前曾在使用前列腺肿瘤和使用正常组织中的超级衍生的区域非编码非编码RNA（UCRNAS）表达超级衍生的非编码RNA（UCRNAS）的表达。我们还发现，某些关键的UCRNA被人类前列腺癌细胞的表观遗传机制沉默。我们的实验室是第一个探索UCRNA在前列腺癌中的全基因组表达谱的人，也是第一个探索这些RNA的调节机制的实验室。但是，除了微阵列以外的其他方法对UCRNA进行量化一直具有挑战性。最近，使用纳米串NCounter分析系统的使用使我们可以通过这种新引入的技术直接量化UCRNA转录本，该技术可能会匹配实时PCR和基于阵列的测定，因为它可以直接和绝对的方式测量RNA。 其他实验表明，UCRNA转录模式可以通过（合成）雄激素暴露和基于siRNA的候选UCRNA下调的下调可以导致全局mRNA表达变化，这表明UCRNA在前列腺癌中可能具有广泛的功能作用。