Figure 1.
Theileria is required to maintain the transformed phenotype of parasitized cells.
(A) The parasite-infected TBL3 cells were grown in the presence or absence of Buparvaquone (+Bup) and cell numbers were monitored by counting live cells following trypan blue exclusion. Buparvaquone had no effect on the proliferation of non-parasitized BL3 cells (average ± standard deviation, n = 3). (B) Representation of flow cytometry analysis indicating the induction of apoptosis (sub-G1 population) in TBL3 cells following treatment with Buparvaquone (open histograms - average ± sd, n = 3). (C) Inhibiting Theileria with Buparvaquone caused apoptosis as monitored by Western blot detection of Caspase-3 activation. The arrow indicates cleaved activated Caspase-3 (n = 3). (D) Parasite-infected TBL3 cells formed colonies when grown in soft agar, in contrast to uninfected parental BL3 cells. This transformed phenotype was reversed by incubating with Buparvaquone (+Bup). The number of colonies per plate (average ± sd, n = 3) and their appearance under the microscope 10 days after plating are shown. (average ± sd, n = 3). ***p<0.001.
Figure 2.
The oncomiR miR-155 is upregulated in cells transformed by Theileria.
(A) We performed miRNA microarray analysis using RNA from parasitized TBL3 cells, treated or not with Buparvaquone for 64 hours. The scatter blot represents the expression of all 459 miRNA data points. Six miRs (highlighted by circles) were down-regulated by more that 2.5-fold (log2) upon Buparvaquone treatment. (B) The Vista plot shows conservation of the BIC gene, comparing bovine to human, mouse or chicken sequences. The miR-155 sequence is identical in the bovine (Bta), human (Hsa) and chicken (Gga) genomes. The square boxes represent the positions of the exons in the human BIC gene. Black arrow represents the Transcriptional start site (TSS). (C) Analysis of miR-155 expression by TaqMan qPCR comparing RNA isolated from Thei, TBL3 or BL3 cells in the presence or absence of Buparvaquone (Bup). Transcript levels in untreated cells are shown relative to the control and normalized against RNU6B mRNA (average ± sd, n = 3). (D) Relative RNA levels of immature pri-miR-155 or pre-miR-155 transcripts in parasitized TBL3 or parental BL3 cells in the presence or absence of Buparvaquone (Bup). Transcript levels in untreated cells are shown relative to the control and normalized against β-actin and B2M mRNA (average ± sd, n = 3). *p<0.05, **p<0.01, ***p<0.001.
Figure 3.
The transcriptional induction of miR-155 in Theileria infected-cells is dependent on AP-1.
(A) Sequence alignment of the proximal promoter sequence of the bovine, human and mouse BIC genes. The highly conserved AP-1 sites and TATA box are highlighted. The TSS is indicated by an arrow. (B) Luciferase reporter analysis of BIC promoter activity in cells infected with Theileria (TBL3 or THEI) or non-infected BL3 cells, with or without Buparvaquone treatment. All experiments were normalized by co-transfection with Renilla constructs (average ± sd, n = 3). (C) The effect of mutating AP-1 (mAP1) or NFκB (mNFκB) binding sites in BIC promoter luciferase assays was tested in TBL3 or THEI cells (average ± sd, n = 3). *p<0.05, **p<0.01.
Figure 4.
Bovine DET1 is a direct target of the miR-155 oncomiR.
(A) Luciferase reporters containing the total 3′UTR of DET1 and TP53INP1 show that Buparvaquone induced Luciferase in parasitized TBL3 cells, but not in parental BL3 cells, and Luciferase induction was lost when the miR-155 target binding site was mutated (mDet1) (average ± sd, n = 3). (B) Luciferase-3′UTR analysis demonstrated that the DET1 and TP53INP1 reporters were suppressed by miR-155 transfection in BL3 cells (left panel) and that a miR-155 Sponge inhibitor induced luciferase activity in TBL3 cells (right panel), dependent on the miR-155 target site (average ± sd, n = 3). (C) Western blot analysis of DET1 and its target protein c-Jun. TBL3 cells showed reduced DET1 protein and elevated c-Jun compared to unparasitized BL3 cells, and the levels were reversed by treatment with Buparvaquone. α Tubulin was used as a loading control. Relative quantification (lower panel) indicates the DET1/Tubulin and c-Jun/Tubulin ratios calculated with Image J software (average ± sd, n = 3). (D) Inhibition by the miR-155 Sponge in TBL3 cells caused upregulation of DET1 protein and decreased c-Jun protein. These effects were reversed by siRNA against DET1. Relative quantification (lower panel) indicates the DET1/Tubulin and c-Jun/Tubulin ratios calculated with Image J software (average ± sd, n = 3). (E) In BL3 cells, the transfection of either miR-155 or siRNA DET1 caused decreased DET1 protein and elevated c-Jun protein levels. Relative quantification (lower panel) indicates the DET1/Tubulin and c-Jun/Tubulin ratios calculated with Image J software (average ± sd, n = 3). *p<0.05, **p<0.01, ***p<0.001.
Figure 5.
miR-155 stabilized c-Jun by inhibiting its proteasomal degradation.
(A) Overexpression of miR-155 or depletion of DET1 in BL3 cells increased the half-life of endogenous c-Jun protein. BL3 cells transiently expressing miR-155 or siDET1 were treated with cycloheximide for the indicated times, followed by immunoblot analysis with a c-Jun antibody and semi-quantification with an αTubulin antibody as a loading control. Relative c-Jun protein levels at time 0 were set as 1 (average ± sd, n = 3). (B) Inhibition by the miR-155 Sponge in TBL3 cells decreased the half-life of endogenous c-Jun. These effects were reversed by siRNA against DET1. TBL3 cells transiently expressing miR-155 Sponge +/− siDET1 were treated with cycloheximide for the indicated times, followed by immunoblot analysis with a c-Jun antibody and semiquantification with α-Tubulin as a loading control. Relative c-Jun levels at time 0 were set as 1 (average ± sd, n = 3). (C) Effect of the miR-155 Sponge on c-Jun protein levels was rescued by treating the proteasome inhibitor MG132. TBL3 cells transiently expressing the miR-155 Sponge were treated with MG132 for 3 h, followed by immunoblot analysis with the c-Jun antibody and semiquantification with α Tubulin as a loading control (average ± sd, n = 3). (D) Overexpression of miR-155 or depletion of DET1 in BL3 cells reduced c-Jun ubiquitination. Transfected cells were treated with MG132 for 3 h, followed by endogenous c-Jun immunoprecipitation and immunoblot analysis with indicated antibodies (average ± sd, n = 3). *p<0.05, **p<0.01, ***p<0.001.
Figure 6.
miR-155 drives a feedback regulatory loop.
(A) miR-155 directly regulates the BIC promoter. In unparasitized BL3 cells, transfection with miR-155, siDET1 or c-Jun led to induction of the BIC-Luciferase reporter. Relative luciferase results are shown compared to control transfections in BL3 cells (dark grey bars) (average ± sd, n = 3). (B) In contrast, in TBL3 cells, inhibiting miR-155 or c-Jun (with plasmids encoding miR-155 Sponge or Dominant Negative DN-c-Jun, respectively) led to reduced BIC-Luciferase reporter activity (average ± sd, n = 3). *p<0.05, **p<0.01, ***p<0.001.
Figure 7.
The miR-155/c-Jun loop is essential for growth and survival.
(A) The colony forming potential of TBL3 cells was markedly reduced by transfection with miR-155 Sponge or Dominant Negative (DN) c-Jun. The inhibitory effect of the miR-155 Sponge could be reversed by co-transfection with siRNA inhibiting DET1, compared to a non-relevant scrambled siRNA control. The photographs are representative of three independent experiments and the average number of colonies per plate are indicated (average ± sd, n = 3). ***p<0.001 (B) Representation of flow cytometry analysis indicating the induction of apoptosis (sub-G1 population) of TBL3 expressed miR-155 Sponge (black histograms) (average ± sd, n = 3). (C) miR-155 expression is essential for TBL3 survival. Transfecting TBL3 cells with the inhibitory miR-155 Sponge caused apoptosis as monitored by Western blot detection of Caspase-3 activation. The arrows indicate cleaved activated Caspase-3 (n = 3). (D) A schematic representation of the miR-155/DET1/c-Jun regulatory loop driving oncomiR addiction. The positive feedback loop is provoked by miR-155 repression of DET1 protein translation and DET1-dependent repression of c-Jun protein stability.