MicroRNAs characterize genetic diversity and drug resistance in pediatric acute lymphoblastic leukemia

ABSTRACT: MicroRNAs (miRNAs) regulate activity of protein-coding genes including those involved in hematopoietic cancers. The goal of the current study was to explore which miRNAs are unique for seven different subtypes of pediatric acute lymphoblastic leukemia (ALL). Therefore, the expression levels of 397 miRNAs (including novel miRNAs) were measured by quantitative RT-PCR in 81 pediatric leukemia cases and 17 normal hematopoietic control cases. Except for BCR-ABL-positive and B-other ALL, all major subtypes i.e. T-ALL, MLL-rearranged, TEL-AML1-positive, E2A-PBX1-positive and hyperdiploid ALL have unique miRNA-signatures that differ from each other and from those in healthy hematopoietic cells. Strikingly, the miRNA signature between TEL-AML1-positive and hyperdiploid cases partly overlapped, which suggests a common underlying biology. Moreover, aberrant downregulation of let-7b (~70-fold) in MLL-rearranged ALL was linked to upregulation of oncoprotein c-Myc (P<0.0001). Besides genetic aberrations, in vitro drug resistance predicts clinical outcome. Resistance to vincristine and daunorubicin was characterized by ~20-fold upregulation of miR-125b, miR-99a and miR-100 (P≤0.002). No discriminative miRNAs were found for prednisolone and only one miRNA was linked to L-asparaginase resistance. Finally we show that the expression levels of 14 miRNAs were --independently of subtype-- associated with clinical outcome in pediatric ALL. We conclude that genetic subtypes and drug resistant leukemic cells display characteristic miRNA signatures in pediatric ALL. Functional studies of discriminative and prognostic important miRNAs may provide new insights into the biology of disease. Experiment type: stem-loop real-time (RT) quantitative PCR (RT-qPCR) Bone marrow and peripheral blood samples were collected from children at newly diagnosis of acute lymphoblastic leukemia (ALL). CD34+ -cells were sorted from G-CSF-stimulated blood cell samples of children with a brain tumor or Wilm’s tumor. Thymocytes were isolated from thymic lobes that were resected from children during surgery for congenital heart disease. RNA from the cell samples was extracted using TRIzol reagents (firma). Only RNA with an RNA Integrity Number (RIN) of ≥ 7.5 as measured by the 2100 Bioanalyzer (Agilent, Amstelveen, the Netherlands) was used as input for the RT-qPCR reactions. All miRNAs were validated with the stem-loop real-time (RT) quantitative PCR (RT-qPCR) technique by using either TaqMan MicroRNA Array MicroRNA arrays (v 1.0, Early Access) or Custom TaqMan MicroRNA Array arrays (Applied Biosystems, Foster City, USA). Values represent ∆Ct in each individual patient for whom the specific subtype, identification number and cellular drug-resistance is shown on top of each column. Drug-resistance was based on median LC50 values (concentration of a drug lethal to 50% of the leukemic cells) that have reported prognostic impact in children with newly diagnosed ALL. Median LC50 values were used to assign patients as sensitive (≤ median LC50) or resistant (> median LC50) to the drug in question. The ∆Ct value was calculated according to the following equation: Ct value of the specific miRNA minus the Ct value of the internal reference. In case of the TaqMan MicroRNA Array MicroRNA arrays the mean Ct values for snoR-13 and snoR-14 were used as reference whereas in case of the Custom TaqMan MicroRNA Array arrays snoR-1 was used. MiRNAs validated by the TaqMan MicroRNA Array MicroRNA arrays are listed together with the part numbers of the corresponding (stem-loop) primers as available by Applied Biosystems, Foster City, USA. Primers for the remaining miRNAs were custom designed by Applied Biosystems. Abbreviations: MLL: MLL-rearranged precursor B-ALL, B-other: precursor B-ALL negative for MLL-rearrangements, TEL-AML1, BCR-ABL, E2A-PBX and hyperdiploidy (> 50 chromosomes). CD34+: normal CD34+-sorted blood progenitor cells, nBM: normal bone marrow.

ORGANISM(S): Homo sapiens

SUBMITTER: Monique Den Boer 

PROVIDER: E-GEOD-23024 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress