Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Science, George Washington University, Washington, DC, USA.
J Transl Med. 2014 Jan 6;12:3. doi: 10.1186/1479-5876-12-3.
Nasopharyngeal carcinoma (NPC) is a solid tumor of the head and neck. Multimodal therapy is highly effective when NPC is detected early. However, due to the location of the tumor and the absence of clinical signs, early detection is difficult, making a biomarker for the early detection of NPC a priority. The dysregulation of small non-coding RNAs (miRNAs) during carcinogenesis is the focus of much current biomarker research. Herein, we examine several miRNA discovery methods using two sample matrices to identify circulating miRNAs (c-miRNAs) associated with NPC.
We tested two miRNA discovery workflows on two sample sources for miRNAs associated with NPC. In the first workflow, we assumed that NPC tumor tissue would be enriched for miRNAs, so we compared miRNA expression in FFPE from NPC cases and controls using microarray and RNA-Seq technologies. Candidate miRNAs from both technologies were verified by qPCR in FFPE and sera from an independent NPC sample set. In a second workflow, we directly interrogated NPC case and control sera by RNA-Seq for c-miRNAs associated with NPC, with candidate c-miRNAs verified by qPCR in the sera from the same independent NPC sample set.
Both microarray and RNA-Seq narrowed the miRNA signature to 1-5% of the known mature human miRNAs. Moreover, these two methods produced similar results when applied to the same sample type (FFPE), with RNA-Seq additionally indicating "unknown" miRNAs associated with NPC. However, we found different miRNA profiles in NPC sera compared to FFPE using RNA-Seq, with the few overlapping miRNAs found to be significantly up-regulated in FFPE significantly down-regulated in sera (and vice versa). Despite the different miRNA profiles found in FFPE and sera, both profiles strongly associated with NPC, providing two potential sources for biomarker signatures for NPC.
We determined that the direct interrogation of sera by RNA-Seq was the most informative method for identifying a c-miRNA signature associated with NPC. We also showed that there are different miRNA expression profiles associated with NPC for tumor tissue and sera. These results reflect on the methods and meaning of miRNA biomarkers for NPC in tissue and peripheral blood.
鼻咽癌（NPC）是头颈部的实体瘤。当 NPC 早期被发现时，多模态治疗非常有效。然而，由于肿瘤的位置和缺乏临床体征，早期检测很困难，因此寻找 NPC 的早期检测生物标志物是当务之急。在癌变过程中小非编码 RNA（miRNA）的失调是当前生物标志物研究的重点。在此，我们使用两种样本基质检查了几种 miRNA 发现方法，以鉴定与 NPC 相关的循环 miRNA（c-miRNA）。
我们在两种样本来源上测试了两种 miRNA 发现工作流程，以寻找与 NPC 相关的 miRNA。在第一个工作流程中，我们假设 NPC 肿瘤组织中 miRNA 会富集，因此我们使用微阵列和 RNA-Seq 技术比较了 NPC 病例和对照的 FFPE 中的 miRNA 表达。来自两种技术的候选 miRNA 通过 qPCR 在 FFPE 和独立 NPC 样本集中的血清中进行验证。在第二个工作流程中，我们直接通过 RNA-Seq 对 NPC 病例和对照的血清进行 c-miRNA 检测，候选 c-miRNA 通过相同独立 NPC 样本集中的血清的 qPCR 进行验证。
微阵列和 RNA-Seq 都将 miRNA 特征缩小到已知成熟人类 miRNA 的 1-5%。此外，当应用于相同的样本类型（FFPE）时，这两种方法产生了相似的结果，RNA-Seq 还指示了与 NPC 相关的“未知”miRNA。然而，我们发现使用 RNA-Seq 时 NPC 血清中的 miRNA 图谱与 FFPE 不同，发现少数重叠的 miRNA 在 FFPE 中显著上调，在血清中显著下调（反之亦然）。尽管在 FFPE 和血清中发现了不同的 miRNA 图谱，但这两种图谱都与 NPC 强烈相关，为 NPC 的生物标志物特征提供了两种潜在的来源。
我们确定直接通过 RNA-Seq 对血清进行检测是鉴定与 NPC 相关的 c-miRNA 特征的最有效方法。我们还表明，肿瘤组织和血清中与 NPC 相关的 miRNA 表达图谱不同。这些结果反映了组织和外周血中 NPC 的 miRNA 生物标志物的方法和意义。
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