Silencing of TIM-3 Expression by miR-326 Affects Apoptosis and Proliferation of Human HL-60 Leukemia Cell Line

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.