Negative regulation of TIM-3 expression in AML cell line (HL-60) using miR-330-5p.
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).


CONCLUSION
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.
['Fooladinezhad|Haniyeh|H|', 'Khanahmad|Hossein|H|', 'Ganjalikhani-Hakemi|Mazdak|M|', 'Doosti|Abbas|A|']
D005434:Flow Cytometry D015973:Gene Expression Regulation, Leukemic D055785:Gene Knockdown Techniques D018922:HL-60 Cells D000072597:Hepatitis A Virus Cellular Receptor 2 D006801:Humans D015470:Leukemia, Myeloid, Acute D035683:MicroRNAs D034622:RNA Interference D020133:Reverse Transcriptase Polymerase Chain Reaction D014162:Transfection
TIM-3 AML HL-60 miR-330-5p molecular medicine