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. 2014 Dec 15;9(12):e115003.
doi: 10.1371/journal.pone.0115003. eCollection 2014.

Identifying and validating a combined mRNA and microRNA signature in response to imatinib treatment in a chronic myeloid leukemia cell line

Affiliations

Identifying and validating a combined mRNA and microRNA signature in response to imatinib treatment in a chronic myeloid leukemia cell line

Steven Bhutra et al. PLoS One. .

Abstract

Imatinib, a targeted tyrosine kinase inhibitor, is the gold standard for managing chronic myeloid leukemia (CML). Despite its wide application, imatinib resistance occurs in 20-30% of individuals with CML. Multiple potential biomarkers have been identified to predict imatinib response; however, the majority of them remain externally uncorroborated. In this study, we set out to systematically identify gene/microRNA (miRNA) whose expression changes are related to imatinib response. Through a Gene Expression Omnibus search, we identified two genome-wide expression datasets that contain expression changes in response to imatinib treatment in a CML cell line (K562): one for mRNA and the other for miRNA. Significantly differentially expressed transcripts/miRNAs post imatinib treatment were identified from both datasets. Three additional filtering criteria were applied 1) miRbase/miRanda predictive algorithm; 2) opposite direction of imatinib effect for genes and miRNAs; and 3) literature support. These criteria narrowed our candidate gene-miRNA to a single pair: IL8 and miR-493-5p. Using PCR we confirmed the significant up-regulation and down-regulation of miR-493-5p and IL8 by imatinib treatment, respectively in K562 cells. In addition, IL8 expression was significantly down-regulated in K562 cells 24 hours after miR-493-5p mimic transfection (p = 0.002). Furthermore, we demonstrated significant cellular growth inhibition after IL8 inhibition through either gene silencing or by over-expression of miR-493-5p (p = 0.0005 and p = 0.001 respectively). The IL8 inhibition also further sensitized K562 cells to imatinib cytotoxicity (p < 0.0001). Our study combined expression changes in transcriptome and miRNA after imatinib exposure to identify a potential gene-miRNA pair that is a critical target in imatinib response. Experimental validation supports the relationships between IL8 and miR-493-5p and between this gene-miRNA pair and imatinib sensitivity in a CML cell line. Our data suggests integrative analysis of multiple omic level data may provide new insight into biomarker discovery as well as mechanisms of imatinib resistance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Work flow for identifying imatinib response biomarkers through an informatics approach in K562 cells.
Figure 2
Figure 2. Relationships among imatinib treatment, and the expression of miR-493-5p and IL8 in K562 cells.
(A) Imatinib treatment (1µM for 24 and 48 hours) significantly up-regulated miR-493-5p expression and significantly down-regulated IL8 expression in K562 cells (*p<0.05, N = 6). (B) IL8 is significantly down-regulated by miR-493-5p mimic at 24 hours after transfection (*p = 0.002, N = 6). Relative fold change was calculated by first normalizing IL8 to B2M and miR-493-5p to RNU6 and then comparing 6 and 24 hours data to 0 hour data.
Figure 3
Figure 3. Biological consequences of IL8 inhibition.
(A) IL8 silencing through siRNA and miR-493-5p mimic resulted in significantly slower cellular proliferation as measured by CellTiter-Glo at 48 hours. (*p<0.05, N = 6). Relative cell growth was calculated by normalizing fluorescence indexes of each time point to the 0 hour control. (B) Cellular response to imatinib treatment curves. Cellular response to imatinib was measured using CellTiter-Glo. p<0.0001 by two way ANOVA, N = 6. Percent survival was calculated by normalizing fluorescence indexes of imatinib treated cells to cells treated with vehicle and plotting on a log(2) scale.

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