title: Negative regulation of TIM-3 expression in AML cell line (HL-60) using miR-330-5p.
abstract: BACKGROUND
  Uncontrolled proliferation and accumulation of leukaemic stem cells (LSCs) in bone marrow leads to acute myeloma leukaemia (AML). T cell immunoglobulin and mucine domain (TIM)-3 is a specific surface marker for LSCs and is highly expressed on LSCs compared with normal bone marrow cells, haematopoietic stem cells. Studies have indicated that microRNAs can affect AML progression through targeting different genes expressions like TIM-3. So, based on bioinformatics assessments, we predicted that miR-330-5p may highly inhibit TIM-3 expression. The purpose of the present study was to prove the silencing effect of miR-330-5p on TIM-3 gene expression in AML cell line (HL-60) in vitro.
  
  
  METHODS
  HL-60 cells were cultured in RPMI 1640 supplied with 10% FBS. TIM-3 expression was induced in the cells using phorbol myristate acetate (PMA). The cells were transfected with miR-330-5p and then, the gene and protein expression of TIM-3 were measured using q-RT-PCR and flow-cytometry methods, respectively.
  
  
  RESULTS
  The results of our bioinformatics surveys revealed that miR-330-5p has high predicted ability to silence TIM-3 gene expression. Accordingly, our experiments confirmed that miR-330-5p is able to strongly silence TIM-3 expression (98.15% silencing) in HL-60 cell line (p = 0.0001).
  
  
  CONCLUSIONS
  According to our results, miR-330-5p has a strong inhibitory effect on TIM-3 expression in AML cell line. Thus, the bioinformatics prediction of Mirwalk and Target Scan softwares for silencing effect of miR-330-5p on TIM-3 is confirmed.
authors.affiliation: a Department of Genetics, Faculty of Sciences, Shahrekord Branch , Islamic Azad University , Shahrekord , Iran.
authors.forename: Haniyeh
authors.initials: H
authors.lastname: Fooladinezhad
authors.affiliation: b Department of Genetics, Faculty of Medicine , Isfahan University of Medical Sciences , Isfahan , Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Khanahmad
authors.affiliation: c Immunology Department, Faculty of Medicine , Isfahan University of Medical Sciences , Isfahan , Iran .
authors.forename: Mazdak
authors.initials: M
authors.lastname: Ganjalikhani-Hakemi
authors.affiliation: a Department of Genetics, Faculty of Sciences, Shahrekord Branch , Islamic Azad University , Shahrekord , Iran.
authors.forename: Abbas
authors.initials: A
authors.lastname: Doosti
mesh_terms: D005434:Flow Cytometry; D015973:Gene Expression Regulation, Leukemic / Q000235:genetics*; D055785:Gene Knockdown Techniques; D018922:HL-60 Cells; D000072597:Hepatitis A Virus Cellular Receptor 2 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: C448908:HAVCR2 protein, human; D000072597:Hepatitis A Virus Cellular Receptor 2; C570805:MIRN330 microRNA, human; D035683:MicroRNAs
keywords: AML; HL-60; TIM-3; miR-330-5p; molecular medicine