Abstract
BackgroundDeregulation 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.MethodsTo 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.ResultsIn 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.ConclusionsRNA editing quantification enables rapid detection of malignant progenitors signifying cancer progression and therapeutic resistance, and will aid future RNA editing inhibitor development efforts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0370-3) contains supplementary material, which is available to authorized users.
Highlights
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)
Eight leukemia stem cells (LSC)-specific loci (Table 1) were selected on the basis of greatest average fold-change in RNA editing frequency among significantly different sites (p < 0.005) [1]. These sites were located within transcripts of the ubiquitin ligase human homolog of mouse double minute 2 (MDM2); the cytidine deaminase apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3D (APOBEC3D); antizyme inhibitor 1 (AZIN1); signal recognition particle 9 kDa (SRP9); splicing factor 3b, subunit 3 (SF3B3); abl-interactor 1 (ABI1); lysosomal trafficking regulator (LYST); and MDM4
Selection and validation of aberrant RNA editing events associated with leukemia progression Previous whole transcriptome analysis (Figure 1A) revealed widespread A-to-I RNA changes in CML LSC that were associated with malignant progenitor reprogramming typified by mis-splicing of key stem cell regulatory transcripts in selective niches during CML progression [1,27]
Summary
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). 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. Deregulation of RNA editing by adenosine deaminases acting on double-stranded (ds) RNA (ADARs) has been implicated in the progression of diverse human malignancies, including chronic and acute forms of leukemia [1,2,3], lobular breast cancer [4], hepatocellular carcinoma [5,6], and esophageal squamous cell carcinoma [7]. ADAR1 plays a key role in hematopoietic stem cell (HSC) survival
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