Important Roles of Cellular MicroRNA miR-155 in Leukemogenesis by Human T-Cell Leukemia Virus Type 1 Infection

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the pathogen that causes the aggressive and lethal malignancy of CD4+ T-lymphocytes called adult T-cell leukemia/lymphoma (ATLL). MicroRNAs (miRNAs), a class of short, noncoding RNAs, regulate gene expression by targeting mRNAs for translational repression or cleavage. miRNAs are involved in many aspects of cell biology linked with formation of several cancer phenotypes. However, the relation between miRNAs and pathologic implication in ATLL is not well elucidated. Here, we evaluated the roles of cellular miRNAs in ATLL caused by HTLV-1. We found that the expression of miR-155 was increased in HTLV-1-positive T-cell lines. miR-155 expression was enhanced by Tax and binding of transcription factors, NF- κ B and AP-1, on the transcription binding sites of miR-155 gene promoter region is important to increase the expression of miR-155 by Tax. Transfection of anti-miR-155 inhibitor, which inhibits the function of miR-155, inhibited the growth of HTLV-1-positive T-cell lines. On the other hand, the growth of HTLV-1-negative T-cell lines was not changed by transfection of anti-miR-155. Forced expression of miR-155 enhanced the growth of HTLV-1-positive T-cell lines. These findings indicate that targeting the functions of miRNAs is a novel approach to the prevention or treatment of ATLL.

Figures

Figure 1

High expression of miR-155 in HTLV-1-positive T-cell lines. The expression of miR-155 in HTLV-1-positive (HTLV-1(+)) and in HTLV-1-negative T-cell lines (HTLV-1(−)) was analyzed by TaqMan real-time RT-PCR. Values are displayed as fold induction of miR-155 expression relative to that in Jurkat cells. Values are the mean ± SD from three separate experiments. Numbers on the MT-2, C5/MJ, and ED-40515(−) represent the real values.

Figure 2

The expression of miR-155 was induced by Tax in T cells. Tax-inducible cell line, JPX-9 cells were cultured with CdCl2 (20 μM) for indicated time periods. (a) Tax expression in CdCl2-treated JPX-9 cells. Cell lysates were prepared from CdCl2-treated JPX-9 cells at the indicated time points. Tax protein expression after CdCl2 treatments was determined by western blot. Actin protein expression served as a loading control. (b) miR-155 expression was analyzed by real-time RT-PCR. miR-155 expression is shown as a fold induction relative to the values measured at 0 h. Values are the mean ± SD from three separate experiments.

Figure 3

miR-155 promoter activity was increased by Tax through both NF-κB and AP-1 activation. (a) Putative transcription factor binding sites in the miR-155 promoter are presented in the left panel. Luciferase reporter plasmids with either wild-type or mutant miR-155 gene promoter together with either Tax expression plasmid (Tax (+)) or empty vector (Tax (−)) were transfected into Jurkat cells. Then the cells were incubated for 48 h. Luciferase reporter activity is shown as a fold induction relative to the levels measured in the cells transfected with the empty vector (Tax (−)). (b) Jurkat cells were transfected with following plasmids: luciferase reporter plasmids of wild-type miR-155 promoter together with either Tax wild type (WT), M22, 703 expression plasmids, or empty vector. Luciferase activity was analyzed 48 h later. Values are the mean ± SD from three separate experiments.

Figure 4

NF-κB signaling is important for miR-155 expression in HTLV-1-positive T cells. (a) DNA-binding activities of proteins to the promoter region of miR-155 gene in HTLV-1-positive and -negative T-cell lines were evaluated by EMSA. The NF-κB oligonucleotide probe containing the NF-κB-binding site from miR-155 gene was used. Arrow shows specific DNA-protein complexes. (b) The specificity of NF-κB-DNA binding was analyzed in MT-2 cells. Competition assay with cold competitors of wild type (WT) or mutated (Mut) probe showed the specificity of the protein-DNA-binding complex. Antibodies to NF-κB subunits were used for super shift assay. The arrow shows specific DNA- NF-κB complexes. The supershifted complexes are indicated by an arrowhead. (c) An NF-κB inhibitor, Bay11-7082, inhibited miR-155 expression. Real-time RT-PCR shows miR-155 expression in MT-2 cells treated with different concentration of Bay11-7082. miR-155 expression is demonstrated as a fold induction relative to that in untreated cells. Values are the mean ± SD from three separate experiments. (d) The activity of NF-κB binding to the miR-155 gene promoter was suppressed by Bay11-7082. MT-2 cells were cultured with 0, 1, 5, or 10 μM of Bay11-7082 for indicated time periods. The NF-κB binding activity on miR-155 gene promoter was determined by EMSA. Specific DNA-NF-κB subunits complexes are indicated by the arrow.

Figure 5

AP-1 signaling is important for miR-155 expression in HTLV-1-positive T cells. (a) DNA-binding activities of proteins to the miR-155 gene promoter in HTLV-1-positive and -negative T-cell lines was assessed by EMSA. The AP-1 oligonucleotide probe containing the AP-1-binding site from miR-155 gene was used. Arrow shows specific DNA-protein complexes. (b) The specificity of AP-1-DNA binding was analyzed in MT-2 cells. Competition assay with cold competitors of wild type probe (WT) or mutated probe (Mut) showed the specificity of the protein-DNA-binding complex. Antibodies against various AP-1 subunits were used for supershift assay. Arrow shows specific complexes of AP-1 with AP-1 probes. Arrowheads show supershifted bands.

Figure 6

The growth of HLTV-1-infected T cells was suppressed by inhibition of cellular miR-155 function. (a) Anti-miR-155 inhibitor (anti-miR-155) or negative control (control) was transfected into the cells (Jurkat, CCRF-CEM, or MT-2 cells). The growth of the cells was determined by trypan blue dye exclusion assay. (b) Pre-miR-155 precursor (pre-miR-155) or negative control (control) was transfected into MT-2 cells. The growth of the cells was determined by trypan blue dye exclusion assay. Mature miR-155 expression at 72 h was determined by TaqMan real-time RT-PCR. Values are the mean ± SD from three separate experiments (*P < 0.05).