Crews Leslie A, Jiang Qingfei, Zipeto Maria A, Lazzari Elisa, Court Angela C, Ali Shawn, Barrett Christian L, Frazer Kelly A, Jamieson Catriona H M
Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center at University of California, La Jolla, CA, 92093, USA.
Sanford Consortium for Regenerative Medicine, La Jolla, CA, 92037, USA.
J Transl Med. 2015 Feb 12;13:52. doi: 10.1186/s12967-014-0370-3.
Deregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populations can be technically challenging, costly and requires PCR validation. The objectives of this study were to validate RNA editing of a subset of cancer stem cell-associated transcripts, and to develop a quantitative RNA editing fingerprint assay for rapid detection of aberrant RNA editing in human malignancies.
To facilitate quantification of cancer stem cell-associated RNA editing in exons and intronic or 3'UTR primate-specific Alu sequences using a sensitive, cost-effective method, we established an in vitro RNA editing model and developed a sensitive RNA editing fingerprint assay that employs a site-specific quantitative PCR (RESSq-PCR) strategy. This assay was validated in a stably-transduced human leukemia cell line, lentiviral-ADAR1 transduced primary hematopoietic stem and progenitor cells, and in primary human chronic myeloid leukemia stem cells.
In lentiviral ADAR1-expressing cells, increased RNA editing of MDM2, APOBEC3D, GLI1 and AZIN1 transcripts was detected by RESSq-PCR with improved sensitivity over sequencing chromatogram analysis. This method accurately detected cancer stem cell-associated RNA editing in primary chronic myeloid leukemia samples, establishing a cancer stem cell-specific RNA editing fingerprint of leukemic transformation that will support clinical development of novel diagnostic tools to predict and prevent cancer progression.
RNA editing quantification enables rapid detection of malignant progenitors signifying cancer progression and therapeutic resistance, and will aid future RNA editing inhibitor development efforts.
作用于双链RNA的腺苷脱氨酶（ADARs）介导的RNA编辑失调与多种人类癌症的进展有关，包括慢性髓性白血病（CML）等血液系统恶性肿瘤。ADAR1的炎症相关激活特异性发生在白血病干细胞中，特别是在CML的晚期，即通常耐药的急变期。然而，通过RNA测序在这些罕见细胞群体中检测癌症干细胞相关的RNA编辑在技术上具有挑战性、成本高昂且需要PCR验证。本研究的目的是验证癌症干细胞相关转录本子集的RNA编辑，并开发一种定量RNA编辑指纹分析方法，用于快速检测人类恶性肿瘤中的异常RNA编辑。
为了使用一种灵敏、经济高效的方法促进对外显子以及内含子或3'UTR灵长类特异性Alu序列中癌症干细胞相关RNA编辑的定量，我们建立了一种体外RNA编辑模型，并开发了一种灵敏的RNA编辑指纹分析方法，该方法采用位点特异性定量PCR（RESSq-PCR）策略。该分析方法在稳定转导的人白血病细胞系、慢病毒-ADAR1转导的原代造血干细胞和祖细胞以及原代人慢性髓性白血病干细胞中进行了验证。
在慢病毒表达ADAR1的细胞中，RESSq-PCR检测到MDM2、APOBEC3D、GLI1和AZIN1转录本的RNA编辑增加，其灵敏度高于测序色谱图分析。该方法准确检测了原代慢性髓性白血病样本中癌症干细胞相关的RNA编辑，建立了白血病转化的癌症干细胞特异性RNA编辑指纹，这将支持预测和预防癌症进展的新型诊断工具的临床开发。
RNA编辑定量能够快速检测预示癌症进展和治疗耐药性的恶性祖细胞，并将有助于未来RNA编辑抑制剂的开发工作。
