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The miR-125a-3p Inhibits TIM-3 Expression in AML Cell Line HL-60 In Vitro.

ABSTRACT: Acute Myeloid Leukemia is a cancer of leukemic stem cells (LSCs) with a rapid progression. It is characterized by overproduction of immature myeloid cells in bone marrow which crowds out normal hematopoietic stem cells (HSC). TIM-3, an immune regulatory molecule, is an LSC specific surface marker in AML with high expression on these cells compared to HSCs. Studies have indicated that micro RNAs (miRNAs) may play an important role in either cancer progression or suppression. Based on bioinformatics assessments, we have predicted that miR-125a-3p could be a miRNA with high suppressive activity on TIM-3 expression. The purpose of this study was to investigate the inhibitory effect of miR-125a-3p on TIM-3 gene expression in an AML cell line, HL-60, in vitro. HL-60 cells were cultured in RPMI 1640 supplied with 10 % FBS. TIM-3 expression was induced on the cells using phorbol miristate acetate. The cells were transfected with miR-125-3p for 24 h and the gene and protein expression of TIM-3 were measured using q-RT-PCR and flow-cytometery methods, respectively. The results of this study showed that miR-125a-3p has a strong silencing effect on TIM-3 gene and protein expression on HL-60 cell line. Based to our results, miR125a-3p can strongly silence TIM-3 expression in AML cell line. Thus, our results have confirmed the bioinformatics prediction of suppressive effect of miR-125a-3p on TIM-3with Mirwalk and Target Scan softwares.

SUBMITTER: Emamdoost F

PROVIDER: S-EPMC5544632 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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The miR-125a-3p Inhibits TIM-3 Expression in AML Cell Line HL-60 In Vitro.

Emamdoost Fatemeh F Khanahmad Hossein H Ganjalikhani-Hakemi Mazdak M Doosti Abbas A

Indian journal of hematology & blood transfusion : an official journal of Indian Society of Hematology and Blood Transfusion 20161006 3

Acute Myeloid Leukemia is a cancer of leukemic stem cells (LSCs) with a rapid progression. It is characterized by overproduction of immature myeloid cells in bone marrow which crowds out normal hematopoietic stem cells (HSC). TIM-3, an immune regulatory molecule, is an LSC specific surface marker in AML with high expression on these cells compared to HSCs. Studies have indicated that micro RNAs (miRNAs) may play an important role in either cancer progression or suppression. Based on bioinformati ...[more]

PMID: 28824235

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Project description:BackgroundT cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a cell surface molecule that was first discovered on T cells. However, recent studies revealed that it is also highly expressed in acute myeloid leukemia (AML) cells and it is related to AML progression. As, Glutamine appears to play a prominent role in malignant tumor progression, especially in their myeloid group, therefore, in this study we aimed to evaluate the relation between TIM-3/Galectin-9 axis and glutamine metabolism in two types of AML cell lines, HL-60 and THP-1.MethodsCell lines were cultured in RPMI 1640 which supplemented with 10% FBS and 1% antibiotics. 24, 48, and 72 h after addition of recombinant Galectin-9 (Gal-9), RT-qPCR analysis, RP-HPLC and gas chromatography techniques were performed to evaluate the expression of glutaminase (GLS), glutamate dehydrogenase (GDH) enzymes, concentration of metabolites; Glutamate (Glu) and alpha-ketoglutarate (α-KG) in glutaminolysis pathway, respectively. Western blotting and MTT assay were used to detect expression of mammalian target of rapamycin complex (mTORC) as signaling factor, GLS protein and cell proliferation rate, respectively.ResultsThe most mRNA expression of GLS and GDH in HL-60 cells was seen at 72 h after Gal-9 treatment (p = 0.001, p = 0.0001) and in THP-1 cell line was observed at 24 h after Gal-9 addition (p = 0.001, p = 0.0001). The most mTORC and GLS protein expression in HL-60 and THP-1 cells was observed at 72 and 24 h after Gal-9 treatment (p = 0.0001), respectively. MTT assay revealed that Gal-9 could promote cell proliferation rate in both cell lines (p = 0.001). Glu concentration in HL-60 and α-KG concentration in both HL-60 (p = 0.03) and THP-1 (p = 0.0001) cell lines had a decreasing trend. But, Glu concentration had an increasing trend in THP-1 cell line (p = 0.0001).ConclusionTaken together, this study suggests TIM-3/Gal-9 interaction could promote glutamine metabolism in HL-60 and THP-1 cells and resulting in AML development.

Project description:BACKGROUND:Recent evidences have shown that circular RNAs (circRNAs) are frequently dysregulated and play paramount roles in various cancers. circRNAs are abundant in central nervous system (CNS); however, few studies describe the clinical significance and role of circRNAs in gliomas, which is the most common and aggressive primary malignant tumor in the CNS. METHODS:A bioinformatics analysis was performed to profile and screen the dyregulated circRNAs during early neural development. Quantitative real-time PCR was used to detect the expression of circ-MAPK4 and target miRNAs. Glioma cells were transfected with circ-MAPK4 siRNAs, then cell proliferation, apoptosis, transwell assays, as well as tumorigenesis and TUNEL assays, were performed to examine effect of circ-MAPK4 in vitro and vivo. Biotinylated-circ-MAPK4 probe based pull-down assay was conducted to confirm the relationship between circ-MAPK4 and miR-125-3p. RESULTS:In this study, we identified a circRNA, circ-MAPK4 (has_circ_0047688), which was downregulated during early neural differentiation. In gliomas, circ-MAPK4 acted as an oncogene, was inversely upregulated and linked to clinical pathological stage of gliomas (P?<?0.05). Next, we verified that circ-MAPK4 promoted the survival and inhibited the apoptosis of glioma cells in vitro and in vivo. Furthermore, we proved that circ-MAPK4 was involved in regulating p38/MAPK pathway, which affected glioma proliferation and apoptosis. Finally, miR-125a-3p, a miRNA exhibited tumor-suppressive function through impairing p38/MAPK pathway, which was increased by inhibiting circ-MAPK4 and could be pulled down by circ-MAPK4. Inhibition of miR-125a-3p could partly rescue the increased phosphorylation levels of p38/MAPK and the elevated amount of apoptosis inducing by knockdown of circ-MAPK4. CONCLUSIONS:Our findings suggest that circ-MAPK4 is a critical player in glioma cell survival and apoptosis via p38/MAPK signaling pathway through modulation of miR-125a-3p, which can serve as a new therapeutic target for treatment of gliomas.

Dataset Information

The miR-125a-3p Inhibits TIM-3 Expression in AML Cell Line HL-60 In Vitro.

Publications

The miR-125a-3p Inhibits TIM-3 Expression in AML Cell Line HL-60 In Vitro.

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