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Silencing of Tim-3 Expression by Mir-326 Affects Apoptosis and Proliferation of Human Hl-60 Leukemia Cell Line Publisher



Mohammadganji M1 ; Ganjalikhanihakemi M1 ; Homayouni V2 ; Rezaei A1 ; Khanahmad H3
Authors
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Authors Affiliations
  1. 1. Isfahan University of Medical Sciences, School of Medicine, Department of Immunology, Isfahan, Iran
  2. 2. Isfahan University of Medical Sciences, Acquired Immunodeficiency Research Center, Isfahan, Iran
  3. 3. Isfahan University of Medical Sciences, School of Medicine, Department of Genetics and Molecular Biology, Isfahan, Iran

Source: UHOD - Uluslararasi Hematoloji-Onkoloji Dergisi Published:2018


Abstract

Leukemia Stem Cells (LSCs) are the main reason for drug-resistance and disease relapse in Acute Myeloid Leukemia (AML). Current drugs destroy normal Hematopoietic Stem Cells (HSCs) rather than LSCs. T cell immunoglobulin mucin-3 (TIM-3), an immune regulatory molecule, is a CD34+CD38- LSCs specific surface marker with high expression on these cells compared to HSCs. The interaction between TIM-3 and its ligand, Galectin-9 (Gal-9), mediates signaling pathways involved in apoptosis and proliferation. We hypothesized that miR-326 could have a suppressive activity on TIM-3 expression and hence, affects the proliferation and apoptosis processes in AML cells. Bioinformatics predictions were done using Mirwalk and Target Scan softwares. TIM-3 expression was induced on HL-60 cells by PMA. After miR-326 transfection, MTT, q-RT-PCR, flow- cytometery were performed to evaluate the cells survival and TIM-3 expression level. Then, after adding recombinant Galectin-9, apoptosis and proliferation rates were measured with Annexin-V and CFSE assays, respectively. Flow cytometry assay confirmed our bioinformatics prediction of suppressive effect of miR- 326 on TIM-3 expression (66.4% silencing) in HL-60 cell line (p= 0.002). The qRT-PCR results were also confirmatory. Annexin-V and MTT assays showed increased cell apoptosis and decreased cell survival, while data from CFSE assay indicated a severe reduction in HL-60 cells proliferation. Our results demonstrated that, miR-326 can silence TIM-3 expression in AML HL-60 cells. Moreover, it is shown that miR-326 can enhance AML cells apoptosis and reduce their proliferation and survival and hence, might be considered as a novel target for therapeutic approaches against AML. © 2018, UHOD - Uluslararasi Hematoloji Onkoloji Dergisi. All rights reserved.
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