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

我们之前曾与俄亥俄州立大学的卡洛·克罗奇博士合作，研究了60例原发人类前列腺癌和16例非肿瘤前列腺癌组织中microRNAs和mRNAs的全基因组表达。分析表明，与周围的非癌组织相比，前列腺癌组织中微RNA加工的关键成分和大量的微RNA都发生了显着变化。与非癌组织相比，肿瘤组织的microRNAs表达上调和下调，肿瘤的表达谱产生了诊断microRNA的特征。值得注意的是，前列腺癌倾向于表达miR-106b-25簇的所有成员，表达水平显著高于非肿瘤前列腺癌，这与miR-106b-25簇在前列腺癌生物学中具有致癌特性是一致的。MiR-1和miR-133在肿瘤中的表达持续低于非肿瘤组织，提示这些microRNAs可能具有肿瘤抑制作用。在对人前列腺癌miR-1-133簇表达的后续研究中，我们在一个独立的数据集中进一步证实了这一发现，并进行了新的观察，发现miR-1的表达在远处转移中进一步降低，并是疾病复发的预测因素。此外，我们还进行了体外实验，以探索miR-1的候选肿瘤抑制功能。基于细胞的分析表明，miR-1在人类前列腺癌细胞中是表观遗传沉默的，对肿瘤的分析表明，在人类肿瘤的一个亚群中，miR-1基因的启动子高甲基化。在这些细胞中过表达miR-1导致生长抑制和调控细胞周期进程、有丝分裂、DNA复制/修复和肌动蛋白动态的基因下调。这一观察结果进一步得到了蛋白质表达分析和基于3-UTR的报告基因分析的证实，表明这些途径中的基因要么是miR-1的直接或间接靶标。基因集浓缩分析显示，miR-1介导的肿瘤抑制作用与组蛋白脱乙酰酶抑制剂的作用总体上相似。最后，我们获得了miR-1改变γ-H_2A.X标志物表达并影响F-肌动蛋白和纤足形成的细胞组织的初步证据。综上所述，我们的研究结果表明miR-1通过影响多个癌症相关过程以及抑制细胞增殖和动力而在前列腺癌中发挥肿瘤抑制作用。我们还继续研究miR-106b-25簇的特定功能。分析来自复发疾病状态的前列腺癌患者的大型microRNA表达数据集，发现miR-106b-25簇在这些肿瘤中表达增加。此外，该簇还被发现在前列腺癌远处转移和TRAMP鼠的前列腺癌中上调。此外，肿瘤miR-106b高表达与早期疾病复发相关，原发灶miR-106b高表达和CASP7(caspase-7)低表达是早期疾病复发的独立预测因素(调整风险比=4.1；95%可信区间：1.6-12.3)。为了确定miR-106b未知的致癌功能，我们在LNCaP人前列腺癌LNCaP细胞中过表达miR-106b，以检测miR-106b诱导的蛋白编码基因的整体表达变化。该方法表明caspase-7是miR-106b的直接靶点，Western印迹分析和3-UTR报告分析证实了这一点。此外，当miR-106b和caspase-7的表达同时被抑制时，由miR-106b基因敲除在DU145人前列腺癌细胞中诱导的某些表型不会发生。进一步的分析表明，caspase-7在多个数据集的原发前列腺癌和转移性病变中下调，并且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可能特异性地影响焦点粘连相关的通路。这一观察是用miR-106b-25转导的22Rv1人前列腺癌细胞进行的实验研究。在感染miR-106b-25慢病毒表达载体后，22Rv1细胞对基底膜和骨基质相关纤维的黏附增加，并促进软琼脂生长。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部黏附相关的途径来实现。我们的发现表明miR-106b-25簇在人类前列腺癌中的作用类似于癌基因，在TRAMP小鼠模型中被概括，并且是早期复发的预测因子。我们先前已经使用微阵列表征了超服务区域衍生非编码RNA(UcRNAs)在前列腺癌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被表观遗传机制沉默。我们的实验室是第一个探索前列腺癌ucRNAs全基因组表达谱的实验室，也是第一个探索这些RNAs调控机制的实验室之一。然而，用其他方法而不是微阵列来定量ucRNAs一直是具有挑战性的。最近，纳米串计数器分析系统的使用使我们能够利用这一新引入的技术直接定量ucRNA转录本，由于其能够以直接和绝对的方式测量RNA，因此可能与定量实时PCR和基于阵列的分析相竞争。其他实验表明，(合成的)雄激素暴露可以改变ucRNA的转录模式，基于siRNA下调候选ucRNAs可以导致全局mRNA表达的变化，表明ucRNAs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.