Expression Profiling of a Panel of Apoptosis Related Micrornas in Patients with Acute Myeloid Leukemia

Introduction: We evaluated the expression of a panel of apoptosis-associated microRNAs (miRNAs) in leukemic blasts isolated from AML patients and investigated their predicted targets.

Patients and Methods: We used bone marrow or peripheral blood that were donated by nine AML patients (5 male, 4 female) at diagnosis. Mononuclear cells were isolated by Ficoll-Histopaque (Sigma Aldrich) density gradient centrifugation and were cryopreserved in liquid nitrogen at the Cancer Biobank Center of the University of Ioannina. MicroBeads technology (Miltenyl Biotec) was used for magnetic cell sorting of CD34+ cells of patients' samples, while mononuclear blood cells from healthy individuals were used as controls. Small RNA (< 200 b) was isolated using the NucleoSpin® miRNA kit (Macherey Nagel). Simultaneous quantification of 84 apoptosis- associated miRNAs was performed by using the miScript miRNA PCR Array Human Apoptosis (MIHS-114ZF, Qiagen) in a LightCycler® 480 instrument (Roche AG, Rotkreuz, Switzerland), and relative quantitation of expression was determined by the comparative CT method. For miRNA target prediction we used the RNA22 tool: https://cm.jefferson.edu/rna22

Results: We found 34 downregulated and 20 upregulated miRNAs compared to control. Among the downregulated miRNAs was the miR-29 family and among the upregulated was the miR-181 family, both of which have been previously implicated in AML. The top 10 downregulated miRNAs were miR-31-5p, miR-451a, miR-29b-3p, miR-409-3p, miR-144-3p, miR-9-5p, miR-192-5p, miR-542-3p, miR-134-5p, miR-29c-3p, whereas the top 10 upregulated miRNAs were miR-34a-5p, miR-181a-5p, miR-181c-5p, miR-181d-5p, miR-181b-5p, miR-222-3p, miR-125b-5p, miR-221-3p, Let-7c-5p, miR-186-3p. We used RNA22 to identify genes that are predicted to be simultaneously targeted by all of the 10 top downregulated miRNAs and also the genes that are predicted to be simultaneously targeted by all of the 10 top upregulated miRNAs. The predicted targets for all top 10 downregulated miRNAs include NSD1, MYST4 and SACS. NSD1 is an histone methyltransferase, whereas MYST4 is an histone acetyltransferase. The predicted targets of all top 10 upregulated miRNAs include 35 genes among which are zinc finger transcription factors with proapoptotic activity (ZNF704, ZNF268) and Solute Carrier (SLC) membrane transporters (SLC24A2, SLC24A4, SLC35E3) of undefined functions. When we compared miRNA expression in patients with abnormal karyotype versus patients with normal karyotype we found miR-125a-5p to be overexpressed in patients with abnormal karyotype. Overexpression of miR-125b has been previously reported in patients with myelodysplastic syndromes and APL and plays important roles in mitochondrial apoptosis.

Conclusions: A variety of microRNAs are dysregylated in patients with AML. We confirm that the miR-29 family and the miR-181 family have altered expression in AML. We found miR-125a-5p to be overexpressed in patients with abnormal karyotype. Among predicted targets of the upregulated miRNAs are genes encoding proapoptotic zinc finger proteins and Solute Carrier membrane transporters, whereas among the predicted targets of the downregulated miRNAs are genes involved in chromatin remodeling, suggesting that altered function of epigenetic modifiers in AML may be due to dysregylation of miRNAs.