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。miR-1和miR-133在肿瘤中的表达始终低于非肿瘤前列腺，表明这些microrna可能具有肿瘤抑制作用。在对人类前列腺肿瘤中miR-1-133簇表达的随访研究中，我们在一个独立的数据集中进一步证实了这一发现，并提出了新的观察结果，即miR-1表达在远处转移中进一步降低，是疾病复发的预测因子。此外，我们还进行了体外实验来探索miR-1的候选肿瘤抑制功能。基于细胞的分析表明，miR-1在人类前列腺癌细胞中是表观遗传沉默的，肿瘤分析表明，miR-1位点在人类肿瘤亚群中启动子超甲基化。miR-1在这些细胞中的过度表达导致生长抑制和调控细胞周期进程、有丝分裂、DNA复制/修复和肌动蛋白动力学的基因下调。蛋白表达分析和基于3- utr的报告基因实验进一步证实了这一观察结果，表明这些通路中的基因是miR-1的直接或间接靶点。一项基因集富集分析显示，mir -1介导的肿瘤抑制作用在全球范围内与组蛋白去乙酰化酶抑制剂相似。最后，我们获得了miR-1改变γ - h2a的初步证据。X标记的表达并影响f -肌动蛋白的细胞组织和足的形成。总之，我们的研究结果表明，miR-1通过影响多种癌症相关过程和抑制细胞增殖和运动，在前列腺癌中发挥肿瘤抑制作用。我们还继续研究miR-106b-25集群的具体功能。对来自复发性前列腺肿瘤患者的大型microRNA表达数据集的分析显示，miR-106b-25簇在这些肿瘤中的表达增加。此外，该簇在前列腺远端转移和前列腺肿瘤小鼠中被发现上调。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报告基因实验得到证实。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的作用类似于致癌基因，这在TRAMP小鼠模型中得到了再现，并且是早期复发的预测因子。我们之前已经使用微阵列表征了超保守区域衍生的非编码rna （ucrna）在前列腺肿瘤和周围正常组织中的表达谱。我们还发现，在人类前列腺癌细胞中，一些关键的ucRNAs通过表观遗传机制被沉默。我们的实验室是第一个探索前列腺癌中ucRNAs全基因组表达谱的实验室，也是第一个探索这些rna调控机制的实验室之一。然而，通过微阵列以外的其他方法定量ucRNAs一直具有挑战性。最近，NanoString nCounter分析系统的使用使我们能够直接定量ucRNA转录物，这种新引入的技术可能与定量实时PCR和基于阵列的分析相媲美，因为它能够以直接和绝对的方式测量rna。其他实验表明，（合成）雄激素暴露可修饰ucRNA转录模式，基于sirna的候选ucRNA下调可导致全局mRNA表达变化，表明ucRNA可能在前列腺癌中具有广泛的功能作用。

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.