Roles for microRNAs, miR-93 and miR-130b, and tumor protein 53-induced nuclear protein 1 tumor suppressor in cell growth dysregulation by human T-cell lymphotrophic virus 1

Abstract

A role for microRNAs (miRNA) in human T-cell leukemia virus 1 (HTLV-1)-mediated cellular transformation has not been described. Here, we profiled miRNA expression in HTLV-1-transformed human T-cell lines and primary peripheral blood mononuclear cells from adult T-cell leukemia patients. Analyses of 11 different profiles revealed six miRNAs that were consistently up-regulated. Two of the up-regulated miRNAs (miR-93 and miR-130b) target the 3' untranslated region (3'UTR) of the mRNA for a tumor suppressor protein, tumor protein 53-induced nuclear protein 1 (TP53INP1). A low expression level of TP53INP1 protein was found in HTLV-1-transformed cells. Additionally, when antagomirs were used to knock down miR-93 and miR-130b in these cells, the expression of TP53INP1 was increased, suggesting that the latter is regulated inside cells by the former. A role for TP53INP1 in regulating cell growth was established by experiments that showed that enhanced TP53INP1 expression increased apoptosis. Collectively, the findings implicate a miR-93/miR-130b-TP53INP1 axis that affects the proliferation and survival of HTLV-1-infected/transformed cells.

Figure 1. Changed miRNA expression in HTLV-1 transformed cell lines, acutely leukemic ATL patient PBMCs, and PMA-activated PBMCs

A) A cell plot analysis of the altered miRNA expression profiles of various HTLV-1 transformed cell lines (MT-1, ATL-55T, ATL-2, ATL-48L, TLOM1, ED, 43T) and acutely leukemic ATL patient PBMCs (Pt1, Pt2, Pt3 and Pt4). The expression patterns are compared with normal PBMCs (PBMC 1, PBMC 2 and PBMC 3). Each colored block represents the expression of one miRNA (labeled on the left) in the indicated sample. Signals acquired from the microarrays are converted into color (high signal = red; low signal = black; no signal = green). Compared with normal PBMCs, elevated (Red colored text) or decreased (blue colored text) expression of miRNAs is indicated. The characterization of the acutely leukemic ATL patient PBMCs (Pt1, Pt2, Pt3 and Pt4) are listed in a tabular format at the bottom. Detectable expression of Tax in the ATL cell lines is tabulated in Supplementary Table 4. B) A Venn analysis of the up-regulated/down-regulated miRNAs in acutely leukemic ATL patient PBMCs (left, yellow shaded) and in ATL-cell lines (right, blue shaded). A subset of miRNAs which are commonly regulated in both cell lines and leukemic PBMCs are shown in the overlapped area. C) A cell plot analysis of the altered miRNA expression profiles of PMA-activated PBMCs. The coloring is the same as described in A. D) MiRNAs common to those up-regulated in all four acutely leukemic ATL PBMCs are highlighted in yellow; those shared with all the ATL-cell lines are highlighted in blue; those shared both with all leukemic PBMCs and ATL-cell lines are highlighted in pink.

Figure 2. Real-time PCR confirmation of the up-regulated miRNAs expression in HTLV-1 transformed cell lines

The HTLV-1 altered miRNAs were verified by quantitative real-time PCR using U6 small RNA as a normalization control. Shown are miR-93, miR-130b, miR-199a* whose level was repressed in ATL cells, and miR30a whose level was not significantly changed in ATL cells. The upper panels show the qRT-PCR profiles and the lower panels show the relative differences (RD) and Ct +/− standard deviations of the samples after normalization to the corresponding miRNAs from normal PBMCs and cord blood cells as described in the text.

Figure 3. miR-93 and miR-130b target the 3′UTR of TP53INP1

A) Schematic representation of miR-93 and miR-130b targets in the 3′UTR of TP53INP1. The positions of the miRNA binding sites correspond to the location of the GenBank sequence NM_033285. B) The 3′UTR of TP53INP1 was PCR-amplified and then cloned downstream a firefly luciferase gene (upper panel). The resulting construct was used to verify the inhibitory activity of miR-93 and miR-130b by transfecting miRNA mimics (miRm-93 and miRm-130b) (lower panel). Specificity of the inhibition was determined by transfecting miRNA mimics with or without the indicated mutations (boxed) in the seed sequences (middle panel). A CMV-driven renilla luciferase construct was co-transfected as a normalization control for firefly luciferase activity.

Figure 4. Induced expression of TP53INP1 by transfection of antagomirs

A) Western blot analysis detected endogenous TP53INP1 expression in Jurkat cells, but not in HTLV-1 transformed cell lines (TLOM1, ED, 43T, C81, MT4 and ATL-2) (Upper panel). Immunoblotting of γ-tubulin was performed as loading controls (Lower panel). B) Tranfection of antagomirs into MT4 cells targeting the cell endogenous miR-93 and miR-130b increased TP53INP1 expression. Immunoblotting of γ-tubulin was performed as a loading control. miR-93 and miR-130b antagomirs are labeled as miR-93i and miR-130bi. Bottom panel shows quantification of the relative intensities of the TP53INP1 bands after normalization to the corresponding tubulin signals. Proliferation and apoptotic assays shown in Figure 5A and B were done in parallel with this experiment using the same set of cell samples (see Figure 5A and B).

Figure 5. Elevated level of TP53INP1 results in reduced cell viability and enhanced apoptosis

A) A modified MTT cell proliferation assay was performed to measure the dehydrogenase activity of viable cells after antagomir transfection. miR-93i- and miR-130bi-transfected cells show dose-dependent lowering of dehydrogenase activity as a measure of viability when compared with control miRNA antagomir cells. B) Cell death of the transfected cells was measured by TUNEL assay which detects DNA strand breakages in apoptotic cells. C) The specificity of a TP53INP1 effect was confirmed by co-transfecting a siRNA against TP53INP1 (siTP) versus an irrelevant control siRNA into miR-93i- and miR-130bi-transfected cells. Viability (left) and cell death (right) were measured. The miR-93i- and miR-130bi-transfected cells were phenotypically rescued by co-transfecting with siTP but not control siRNA. D) Knockdown of TP53INP1 protein in Jurkat cells by transfecting miR-93 and miR-130b mimics enhanced relative cell growth. Top panel shows Western blotting results of TP53INP1 comparing wild type miRm-93 and miRm-130b to mutated miR-93 and mutated miR-130b. Immunoblotting of γ-tubulin was performed as a loading control. Bottom panel shows relative proliferation with the mutant miRNA transfected cells set to 100%.

Figure 6. Evidence that expression of miR-93 and miR-130b is regulated by HTLV-1 infection

A) A schematic representation of the genomic arrangement of miR-130b. The putative miR-130b promoter is shown with the indicated transcription factor binding sites. Regions highlighted in red (in panels A and C) represent the mature miRNAs. B) Transcriptional activity of the miR-130b promoter is activated by Tax. Increasing amounts of Tax transfected into HeLa cells together with the putative miR-130 promoter-driven luciferase vector increased activity (see also Supplementary Figure 1). miR-130b-promoter-less luciferase reporter (pGL3) was used as a control which showed minimal luciferase activity. C) MiR-93 is found within the intron of MCM7. D) The expression levels of MCM7 were determined in the indicated HTLV-1 transformed (MT1, C81, MT4, 43T, ATL2) and non-HTLV-1 transformed (CEM, CEMSS, Jurkat) cell lines (lower panel). Higher levels of MCM7 were found in the HTLV-1 transformed cell lines when compared to the non-HTLV-1 transformed cell lines. The expression level of MCM7 was normalized to the amount of γ-tubulin and plotted in the histogram (upper panel). * and ** represent P < 0.05 and < 0.01 values, when compared with Jurkat cells.