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. 2016 Nov 29;7(48):78667-78679.
doi: 10.18632/oncotarget.12018.

miR-125b regulates differentiation and metabolic reprogramming of T cell acute lymphoblastic leukemia by directly targeting A20

Zixing Liu  1 , Kelly R Smith  1 , Hung T Khong  2 , Jingshan Huang  3 , Eun-Young Erin Ahn  1 , Ming Zhou  4 , Ming Tan  1   5
Affiliations

miR-125b regulates differentiation and metabolic reprogramming of T cell acute lymphoblastic leukemia by directly targeting A20

Zixing Liu et al. Oncotarget. .

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy. Although it has been reported that overexpression of miR-125b leads to T-ALL development, the underlying mechanisms of miR-125b action are still unclear. The goal of this study is to delineate the role of miR-125b in T-ALL development. We found that miR-125b is highly expressed in undifferentiated leukemic T cells (CD4-negative) while its expression is low in differentiated T cells (CD4-positive). Overexpression of miR-125b increased the CD4-negative population in T cells, whereas depletion of miR-125b by miR-125b-sponge decreased the CD4-negative cell population. We identified that A20 (TNFAIP3) is a direct target of miR-125b in T cells. Overexpression of miR-125b also increased glucose uptake and oxygen consumption in T cells through targeting A20. Furthermore, restoration of A20 in miR-125b-overexpressing cells decreased the CD4-negative population in T cell leukemia, and decreased glucose uptake and oxygen consumption to the basal level of T cells transfected with vector. In conclusion, our data demonstrate that miR-125b regulates differentiation and reprogramming of T cell glucose metabolism via targeting A20. Since both de-differentiation and dysregulated glucose metabolism contribute to the development of T-cell leukemia, these findings provide novel insights into the understanding and treatment of T-ALL.

Keywords: A20; T cell; differentiation; lymphocytic leukemia; miR-125b.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. miR-125b regulates T cell differentiation
A. qRT-PCR was performed to examine the expression of miR-125 in CD4+population and CD4-pouplation of Jurkat cell lines. RNU6B was used as an internal control and for normalization of the data. Columns represent the mean of three independent experiments; bars represent SE. ***, p<0.001. B and C. 1×106 Jurkat-vector, Jurkat-miR-125b, T2-vector and T2-miR-125b stable cell lines were incubated with CD4 and CD8 antibodies for 45min, washed, and then the cells were analyzed by flow cytometry.
Figure 2
Figure 2. A20 is a direct target of miR-125b in T cells
A. qRT-PCR was performed to examine the expression of A20 in CD4+population and CD4-pouplation of Jurkat cell lines. GAPDH was used as an internal control and for normalization of the data. Columns represent the mean of three independent experiments; bars represent SE. ***, p<0.001. B and C. Jurkat-vector, Jurkat-miR-125b, T2-vector, T2-miR-125b, HMLE-vector, HMLE-miR-125, Jurkat-scramble and Jurkat-miR-125b-spong1 stable cell lines were collected. Cell lysates were prepared for Western blotting with an antibody against A20 with β-actin used as a loading control. D. A20 3′ UTR contains a predicted miR-125b-binding site. Alignment between the miR-125b seed sequence and A20 3′ UTR is shown. A schematic diagram shows the wild type and mutant 3′UTR of A20. E. Jurkat cells were co-transfected with luciferase reporter plasmids with or without 3′-UTR of A20, pre-miR-125, or pre-miR-negative (Ctr). 48 hours post-transfection, cells were harvested and lysed with lysis buffer. Luciferase activity was measured by using a dual luciferase reporter assay. The pRL-TK vector was used as an internal control. The results were presented as relative luciferase activity (firefly LUC/Renilla LUC).
Figure 3
Figure 3. MiR-125 regulates CD4 differentiation marker in T-ALL cells through targeting A20
A. 1×106 Jurkat-vector and Jurkat-A20 stable cell lines were incubated with CD4 and CD8 for 45min, washed, and then the cells were analyzed by flow cytometry. Jurkat-vector and Jurkat-A20 stable cell lines were collected. Cell lysates were prepared for Western blotting with an antibody against A20, and β-actin was used as a loading control. B. Jurkat cells were transfected with scrambled siRNA or A20 siRNAs. Forty-eight hrs after transfection, Cells were harvested and used for extracting RNA. qRT-PCR was performed to validate the expression of CD4 in Jurkat with depletion of A20 by siRNA. GAPDH was used as an internal control and for normalization of the data. Columns represent the mean of three independent experiments; bars represent SE. ***, p<0.001. C and D. 1×106 Jurkat-vector and Jurkat-miR-125b, Jurkat-miR-125b transfected with A20, T2 vector, T2-miR-125b, T2-miR-125b transfected with A20 cell lines were incubated with CD4 and CD8 antibodies for 45min, washed, and then the cells were analyzed by flow cytometry. bars represent SE. **, p<0.01.
Figure 4
Figure 4. miR-125b inhibition of the differentiation of T cells is NF-kB independent
A. Jurkat-vector and Jurkat-miR-125b cells were transfected with NF-κB reporter by using Lipofectamine 2000 reagent. 48 hours post-transfection, cells were harvested and lysed with passive lysis buffer. Luciferase activity was measured by using a dual luciferase reporter assay. The pRL-TK vector was used as an internal control. The results were expressed as relative luciferase activity (firefly LUC/Renilla LUC). B. Jurkat-vector, Jurkat-A20, T2-vector, T2 –miR-125b, HMLE-vector and HMLE-miR-125b stable cell lines were collected. Cell lysates were prepared for Western blotting with antibodies against p65 and p65-p. β-actin was used as a loading control. C and D. Jurkat-miR-125b and T2-miR-125b cells were treated with DMSO (control) and NF-κB inhibitors (Calbiochem) for 48 hrs. Cells were incubated with CD4 and CD8 antibodies for 45 min, washed, and then the cells were analyzed by flow cytometry.
Figure 5
Figure 5. miR-125b regulates the glucose metabolism in T cells via A20
A and D. Cells were cultured in medium containing 10% fetal bovine serum (FBS) and glucose uptake was measured. Data are showing percentage relative to Jurkat –vector (left) or T2 vector (right). B and E. Oxygen consumption rates of Jurakt-vector, Jurkat-miR-125b, Jurkat-miR-125b transfected with A20 (left), T2-vector and T2-miR-125b Jurkat-miR-125b transfected with A20 cells (right) were measured. The oxygen consumption rate was calculated on the basis of the maximal rate of change in relative fluorescence units (DFU/second). Columns, mean of three independent experiments C. Cells were cultured in medium containing10%FBS and lactate production in the medium was measured as described in Materials and methods. Data are showing percentage. Jurkat-vector, Jurkat-A20, T2-vector and T2 –miR-125b stable cell lines were collected. Cell lysates were prepared for Western blotting with antibodies against PDK1 and LDHA. β-actin was used as a loading control.
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