Research Article: Differential expression of miRNAs in acute myeloid leukemia quantified by Nextgen sequencing of whole blood samples

Date Published: March 20, 2019

Publisher: Public Library of Science

Author(s): Aakriti Pandita, Poornima Ramadas, Aarati Poudel, Nibal Saad, Ankit Anand, Alina Basnet, Dongliang Wang, Frank Middleton, Diana M. Gilligan, Francesco Bertolini.


New approaches are needed for understanding and treating acute myeloid leukemia (AML). MicroRNAs (miRs) are important regulators of gene expression in all cells and disruption of their normal expression can lead to changes in phenotype of a cell, in particular the emergence of a leukemic clone. We collected peripheral blood samples from 10 adult patients with newly diagnosed AML, prior to induction chemotherapy, and 9 controls. Two and a half ml of whole blood was collected in Paxgene RNA tubes. MiRNA was purified using RNeasy mini column (Qiagen). We sequenced approximately 1000 miRs from each of 10 AML patients and 9 controls. In subset analysis, patients with NPM1 and FLT3 mutations showed the greatest number of miRNAs (63) with expression levels that differed from control with adjusted p-value of 0.05 or less. Some of these miRs have been described previously in association with leukemia, but many are new. Our approach of global sequencing of miRs as opposed to microarray analysis removes the bias regarding which miRs to assay and has demonstrated discovery of new associations of miRs with AML. Another strength of our approach is that sequencing miRs is specific for the 5p or 3p strand of the gene, greatly narrowing the proposed target genes to study further. Our study provides new information about the molecular changes that lead to evolution of the leukemic clone and offers new possibilities for monitoring relapse and developing new treatment strategies.

Partial Text

One area of research that has grown explosively in the past 15 years is the study of non-coding RNA. MiRNAs are 19–22 nucleotide long non-coding RNAs which regulate the expression of genes by sequence-specific binding to mRNA to either promote or block its translation [1]. This is a powerful level of epigenetic control for gene expression that can influence the phenotype of a cell [2]. Several authors have examined the role of miRNA in the transformation of hematopoietic stem cells into leukemic cells [3–8]. It is now well established that miRNAs play a role in blocking differentiation of leukemic cells and promoting their unchecked cell division [9,10].

This protocol was reviewed and approved by the Upstate Medical University Institutional Review Board. Blood was obtained following written informed consent from patients at University Hospital with newly diagnosed AML, prior to start of induction chemotherapy. Blood was also obtained following written informed consent from healthy volunteers involved in patient care at Upstate. 2.5 ml of whole blood was collected into Paxgene RNA preparation tubes and stored at -80C for batch processing. miRNA was purified using a Qiagen miRNA purification kit. The yield and quality of the RNA samples was assessed using the Agilent Bioanalyzer prior to library construction using the Illumina TruSeq Small RNA Sample Prep protocol (Illumina; San Diego, California). Multiplexed samples of RNA that exceed quality control metrics (RIN > 6.0) were run on an Illumina NextSeq500 instrument at a targeted depth of 10 million reads per sample. After filtering and trimming of index and adapter sequences, whole genome alignment of the miR FASTQ reads was performed using the Homo sapiens/hg21 reference genome in the SHRiMPS aligner included in the miRNAs analysis application available in BaseSpace (Illumina), as well as the sRNA Toolbox application suite.

We sequenced all miRNAs in peripheral whole blood from ten patients with newly diagnosed AML and nine normal controls. Table 1 shows the characteristics of the ten patients who entered the study. There were five males and five females, ages ranged from 42 to 87. Initial white blood cell (WBC) count ranged from 1.1 x 103/ul to 88 x 103/ul, with blast percentage of 3.8 to 73. Phenotype was determined by standard hematopathology staining for surface markers. For all ten patients, we recorded presence (pos) or absence (neg) of NPM1 or FLT3 mutations and cytogenetic analysis.

Rather than analyze by microarray which would identify only 300–400 miRNAs, we chose to sequence all miRNAs that were obtained in whole blood samples from patients with newly diagnosed AML and controls. We quantified expression of 996 miRNAs from each patient and control and performed statistical analyses to determine which miRNAs were increased or decreased in AML patients versus normal controls. Subset analysis revealed the most differences in patients with double positive AML (NPM1+/FLT3+ mutations). NPM1 mutations are the most common genetic abnormalities in AML (50–60% of cytogenetically normal AML and 30% of all AML) [15]. Up to one-third of NPM1+ patients also have a mutation in FLT3, which counteracts the favorable prognosis of the NPM1 mutation [16]. The NPM1 gene encodes a 32-kDA protein that is involved in numerous cellular processes. It can function as an oncogene and a tumor suppressor gene depending on expression levels, interacting proteins, and cellular compartmentalization. Dozens of mutations have been described in NPM1 and how they contribute to leukemogenesis is not known. Finding 63 distinct miRNAs either upregulated or downregulated in NPM1+ /FLT3+ AML suggests that there may be multiple molecular pathways disrupted that contribute to the leukemic phenotype.




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