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Evaluation of Silencing Effect of Mir-133A-5P Mimic on Tim-3 Expression in Aml (Hl-60) Cell Line Publisher



Hojati Z1 ; Ganjalikhanihakemi M2 ; Ameri M1 ; Alimohammadijelodar SF1 ; Dehbashi M1 ; Mohammad Ganji M2 ; Homayouni V3 ; Khanahmad H4
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Authors Affiliations
  1. 1. Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, 81746-73441, Iran
  2. 2. Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3. Acquired Immunodeficiency Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
  4. 4. Department of Genetic and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Source: Indian Journal of Clinical Biochemistry Published:2020


Abstract

Acute myelogenous leukemia (AML) is a complex blood malignancy leading to immature leukemic stem cells (LSCs) proliferation. T cell immunoglobulin mucin-3 (TIM-3) is known as a biomarker of AML LSCs. Several microRNAs (miRNAs) can affect gene expression in AML. In this study, the silencing effect of miR-133a-5p on TIM-3 expression in AML cell lineage (HL-60) was investigated. It’s been hypothesized that miR-133a-5p may suppress the TIM-3 expression in AML cell line. Initially, miRNA-TIM-3 prediction, enrichment, and network analysis were done. Then, miR-133a-5p mimic was transfected into HL-60 cells. The TIM-3 protein and gene expression were measured by flow cytometry analysis and real-time PCR, respectively. MTT assay was also carried out. Based on the Bioinformatics predictions, miR-133a-5p was able to silence TIM-3 expression. Also, significant pathways pertained to miR-133a-5p were obtained using enrichment analysis. According to this, miR-133a-5p was mainly engaged in the MAPK signaling pathway and Nicotine addiction pathway using the KEGG database. The TIM-3 protein expression of the transfected cells was measured as 17.15 ± 8.87% (p = 0.001). A 52.48% significant gene silencing in mRNA level was obtained in comparison to the negative control. Despite of down regulation of TIM-3, HL-60 cell viability has not been significantly changed. It has been finally confirmed that miR-133a-5p could strongly suppress TIM-3 expression in AML cell line. Presumably, down regulation of TIM-3 could affect MAPK and Nicotine addiction signaling pathways. © 2019, Association of Clinical Biochemists of India.
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