MicroRNA-125b transforms myeloid cell lines by repressing multiple mRNA - PubMed (original) (raw)

MicroRNA-125b transforms myeloid cell lines by repressing multiple mRNA

Marina Bousquet et al. Haematologica. 2012 Nov.

Abstract

Background: We previously described a t(2;11)(p21;q23) chromosomal translocation found in patients with myelodysplasia or acute myeloid leukemia that leads to over-expression of the microRNA miR-125b, and we showed that transplantation of mice with murine stem/progenitor cells overexpressing miR-125b is able to induce leukemia. In this study, we investigated the mechanism of myeloid transformation by miR-125b.

Design and methods: To investigate the consequences of miR-125b over-expression on myeloid differentiation, apoptosis and proliferation, we used the NB4 and HL60 human promyelocytic cell lines and the 32Dclone3 murine promyelocytic cell line. To test whether miR-125b is able to transform myeloid cells, we used the non-tumorigenic and interleukin-3-dependent 32Dclone3 cell line over-expressing miR-125b, in xenograft experiments in nude mice and in conditions of interleukin-3 deprivation. To identify new miR-125b targets, we compared, by RNA-sequencing, the transcriptome of cell lines that do or do not over-express miR-125b.

Results: We showed that miR-125b over-expression blocks apoptosis and myeloid differentiation and enhances proliferation in both species. More importantly, we demonstrated that miR-125b is able to transform the 32Dclone3 cell line by conferring growth independence from interleukin-3; xenograft experiments showed that these cells form tumors in nude mice. Using RNA-sequencing and quantitative real-time polymerase chain reaction experiments, we identified multiple miR-125b targets. We demonstrated that ABTB1, an anti-proliferative factor, is a new direct target of miR-125b and we confirmed that CBFB, a transcription factor involved in hematopoiesis, is also targeted by miR-125b. MiR-125b controls apoptosis by down-regulating genes involved in the p53 pathway including BAK1 and TP53INP1.

Conclusions: This study demonstrates that in a myeloid context, miR-125b is an oncomiR able to transform cell lines. miR-125b blocks myeloid differentiation in part by targeting CBFB, blocks apoptosis through down-regulation of multiple genes involved in the p53 pathway, and confers a proliferative advantage to human and mouse myeloid cell lines in part by targeting ABTB1.

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Figures

Figure 1.

Figure 1.

miR-125b blocks apoptosis of mouse and human cell lines (A) 32Dclone3 infected cells were deprived of IL-3 from the media and annexinV-phycoerythrin/7-aminoactinomycin D (7-AAD) staining was performed 4 days later. Annexin V+/7-AAD- cells are apoptotic cells. One representative flow cytometry plot is shown. The histogram represents the average of apoptotic cells (annexin V+) from three independent experiments. (B) The human promyelocytic NB4 cell line was transiently transfected with a miR-125b mimic or mimic control. One day later, apoptosis was induced by adding camptothecin and the percentage of apoptotic cells was assessed 2 days later by annexin V/7-AAD staining. One representative flow cytometry plot is shown. The histogram represents the average of apoptotic cells (annexin V+) from five independent experiments. (C) The human promyelocytic HL60E cell line was stably infected with XZ or XZ-miR-125b. GFP+ cells were sorted and induced to apoptosis by camptothecin treatment. Apoptosis was quantified 2 days later by flow cytometry with annexin V staining. One representative flow cytometry plot is shown. The histogram represents the average of apoptotic cells (annexin V+) from three independent experiments.

Figure 2.

Figure 2.

miR-125b confers a proliferative advantage to human and mouse myeloid cells. (A) 32Dclone3 infected cells (GFP+) expressing or not miR-125b were mixed with 32Dclone3 wild-type cells (GFP–) at a ratio ∼1:3. The percent of GFP+ cells in the population was determined every 3 days of culture. Data represent the average of four independent experiments done with two different batches of virus for each condition. (B) The same experiment was performed with HL60E infected cells.

Figure 3.

Figure 3.

miR-125b is an oncomiR able to transform 32Dclone3 cells by conferring independence to growth factor removal and inducing tumors in nude mice (A) Independence from growth factors was assessed by removal of IL-3 from the 32Dclone3 media. Cells were resuspended at 300 000 cells/mL every 3 days. Viability of the cells was determined by flow cytometry analysis with annexin V/7-aminoactinomycin D (7-AAD) staining every 2 or 3 days. Annexin V–/7-AAD– cells are live cells. Data represent the average of three independent experiments. (B) 1×107 32Dclone3 cells over-expressing miR-125b (n=30 total) or control (n=20 total) 32Dclone3 cells were subcutaneously injected into the backs of nude mice. Xenograft engraftment was monitored weekly and tumor size was recorded. Mice were sacrificed when the tumor reached 1 cm in diameter. Experiments were performed twice with 15 and 10 mice respectively injected with miR-125b overexpressing cells or control 32Dclone3 cells. (C) Pictures of nude mouse injected with miR-125b 32Dclone3 cells showing the tumor (left), hepatomegaly and splenomegaly (middle) and infiltrated lymph nodes (right).

Figure 4.

Figure 4.

Identification of miR-125b targets: ABTB1 is a miR-125b target (A) Quantitative reverse transcriptase-PCR of ABTB1 mRNA in 32Dclone3 cells overexpressing miR-125b compared to 32Dclone3 control. *P<0.0005. (B) Western blot showing the down-regulation of ABTB1 protein in miR-125b over-expressing cells compared to control cells (upper panel). ABTB1 is increased in 32Dclone3 cells transiently transfected with an inhibitor of miR-125b (lower panel). (C) Repression of luciferase activity due to the binding of miR-125b to the 3'UTR of ABTB1. The 3'UTR of ABTB1 containing the predicted binding site for miR-125b was cloned 3' to the renilla luciferase open reading frame in the psicheck2 vector. The ABTB1 3'UTR mut corresponds to the same construct with an internal mutation in the binding site of miR-125b. chek2 is the empty vector and it serves as the negative control. The perfect match construct is the positive control containing the miR-125b binding site only. Each construct was co-transfected in 293T cells with miR-125b mimics or control mimics and luciferase activity was assessed 2 days after transfection. Renilla activity was normalized to the firefly internal psicheck control. The results presented correspond to the relative luciferase activity normalized to transfections with control mimics. *P<0.0005. (D) miR-125b targets genes involved in apoptosis are down-regulated by miR-125b expression in both human and mouse myeloid cell lines. The mRNA expression levels of BAK1, PLAGL1, PLK3, PPP1CA, PPP2CA and TP53INP1 were measured in 32Dclone3 and HL60 cells infected with XZ (control) or XZ-miR-125b vectors, and in NB4 cells 3 days after transient transfection with control or miR-125b mimics. The mRNA expression levels were evaluated by quantitative real-time PCR, normalized to the expressions of MLN51 and ACTIN in human cells and GAPDH in mouse cells, and presented as fold change [2-ΔΔCt] ± SD (n ≥ 3) in miR-125b expressing cells relative to control cells. Two-tailed t-test results of *P<0.05 relative to control cells.

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