Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice

Abstract

We showed that Emicro-MiR-155 transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre-B-cell proliferation, have variable clinical presentation, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain-containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein beta (C/EBPbeta), 2 important regulators of the interleukin-6 signaling pathway, are direct targets of MiR-155 and become gradually more down-regulated in the leukemic than in the preleukemic mice. We hypothesize that miR-155, by down-modulating Ship and C/EBPbeta, initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma.

Figure 1

Comparative Kaplan Meier survival curve for MiR-155 transgenic mice. (A) Survival of FVB and C57BL/6 MiR-155 transgenics compared with their wild-type counterparts, showing shorter lifespan for the transgenic mice. (B) Survival of transplanted FVB mice compared with the wild-type controls; all transplanted mice were dead after 6 weeks, whereas all the wild-type controls were alive.

Figure 2

MiR-155 preleukemic transgenic mice are characterized exclusively by an expansion of the pre–B-cell population, whereas the MiR-155–induced leukemias display a mixed B220^low/IgM⁻ and B220^low/IgM⁺ immunophenotype. The myeloid lineage was equally expanded in all transgenics, preleukemic and leukemic alike. (A) Flow cytometry for B220 and IgM shows an increase of the B220⁺/IgM⁻ population in the preleukemic spleens compared with the wild-type ones; the leukemic spleens exhibit a mixed B220^low/IgM⁺ and B220^low/IgM⁻ immunophenotype. (B) Flow cytometry for CD11b shows an important increase of the myeloid lineage in the transgenic spleens (preleukemic and leukemic) compared with the wild-types.

Figure 3

MiR-155 transgene is differentially expressed in various hematopoietic organs and B-cell subpopulations, as assessed by TaqMan assay. (A) TaqMan assay showed that MiR-155 has the highest expression in the bone marrow (BM = bone marrow; Liv = liver; Sp = spleen; Thy = thymus). Note: Tg1 belongs to the founding line F8, whereas tg2 belongs to the founding line F10. (B) TaqMan assay indicated that MiR-155 transgene is much more expressed in the pre-B cells than in the mature B ones (TgDbl = transgenic mature B cells; TgPE = transgenic pre-B cells; wtDbl = wild-type mature B cells; wtPE = wild-type pre-B cells). Note: Cells were sorted from transgenics belonging to the same founding line, F8.

Figure 4

Ship and C/ebpβ are direct targets of miR-155. (A) Spleen, immunohistochemistry, 200×. SHIP-1 is highly expressed in normal B and T lymphocytes (Olympus BX41 microscope and software; the bar represents 200 μm). (B) Malignant lymphocytes do not stain for SHIP-1 due to the loss of its expression. (C-D) Luciferase activity assay for Ship-3′-UTR and C/ebpβ, wild-type and mutant, after the control (□) and miR-155 (■) transfections in 293 cells.

Figure 5

The Ship and C/ebpβ down-regulation in the miR-155 transgenic splenocytes compared with the wild-type splenocytes occurs mainly in the B-cell precursors rather than in the mature B cells. (A-B) Western blots on proteins extracted from total splenocytes show the lack of expression of SHIP-1 (A) and C/EBPβ (B) in the leukemic compared with the wild-types. Note: The samples marked “T” are 2 T-cell leukemias arisen in the miR-155 transgenic mice.

Figure 6

Western blots on flow cytometry-sorted B cells for SHIP and C/ebpβ show that miR-155 leukemic pre-B cells have lower expression of these 2 proteins compared with the wild-type counterparts. (A) Immunoblot shows a stepwise down-regulation of the SHIP expression in the pre-B and mature B cells in the leukemic sorted cells compared with their preleukemic and wild-type counterparts. (B) Densitometry for SHIP expression in pre-B cells calculated on the previous immunoblot confirms that SHIP is expressed the most in the wild-type, less in the preleukemic, and the least in the leukemic cells. (C) Immunoblot on sorted splenocytes shows a down-regulation of C/ebpβ in the pre-B cells of leukemic mice compared with their preleukemic and wild-type counterparts. (D) Densitometry for the C/ebpβ expression in different pre-B cells (wild-type, preleukemic, and leukemic), calculated on the previous immunoblot, confirms that C/ebpβ has higher expression in the leukemic pre-B cells than in the preleukemic and wild-type ones.

Figure 7

Schematic presentation of the proposed IL-6–SHIP–C/EBPβ–MiR-155 interactions in B cells, and their role in lineage differentiation.