MicroRNA-21 plays an oncogenic role by targeting FOXO1 and activating the PI3K/AKT pathway in diffuse large B-cell lymphoma

Abstract

The prognostic implications of miR-21, miR-17-92 and miR-155 were evaluated in diffuse large B-cell lymphoma (DLBCL) patients, and novel mechanism by which miR-21 contributes to the oncogenesis of DLBCL by regulating FOXO1 and PI3K/AKT/mTOR pathway was investigated. The expressions of miR-21, miR-17-92 and miR-155 measured by quantitative reverse-transcription-PCR were significantly up-regulated in DLBCL tissues (n=200) compared to control tonsils (P=0.012, P=0.001 and P<0.0001). Overexpression of miR-21 and miR-17-92 was significantly associated with shorter progression-free survival (P=0.003 and P=0.014) and overall survival (P=0.004 and P=0.012). High miR-21 was an independent prognostic factor in DLBCL patients treated with rituximab-combined chemotherapy. MiR-21 level was inversely correlated with the levels of FOXO1 and PTEN in DLBCL cell lines. Reporter-gene assay showed that miR-21 directly targeted and suppressed the FOXO1 expression, and subsequently inhibited Bim transcription in DLBCL cells. MiR-21 also down-regulated PTEN expression and consequently activated the PI3K/AKT/mTOR pathway, which further decreased FOXO1 expression. Moreover, miR-21 inhibitor suppressed the expression and activity of MDR1, thereby sensitizing DLBCL cells to doxorubicin. These data demonstrated that miR-21 plays an important oncogenic role in DLBCL by modulating the PI3K/AKT/mTOR/FOXO1 pathway at multiple levels resulting in strong prognostic implication. Therefore, targeting miR-21 may have therapeutic relevance in DLBCL.

Keywords: FOXO1; diffuse large B-cell lymphoma; miR-155; miR-17-92 cluster; miR-21.

Figure 1. Expression of miR-21, the miR-17-92 cluster and miR-155 in tumor tissues of DLBCL patients and their prognostic implications

A, The levels of expression of miR-21, the miR-17-92 cluster and miR-155 were evaluated in the tumor tissues of DLBCL patients (n = 200) and in normal tonsils as a control (n = 11) using qRT-PCR. Box-and-whisker plots demonstrate the levels of expression of miR-21, the miR-17-92 cluster, and miR-155 as dCt (= Ct_(miRNA) - Ct_(U6)) values in the DLBCL tissues compared with those in normal tonsils. A lower dCt implies a higher relative level of miRNA. The differences in the miRNA levels of the DLBCL and normal group were evaluated using Student's t-test. B, The relative expression levels of the miRNAs were calculated as ddCt (= dCt_(case) - mean dCt_(control)). The diagrams show the correlation of the relative expression levels of miR-21, the miR-17-92 cluster and miR-155 determined using Pearson's correlation analysis (r, correlation coefficient). Kaplan-Meier curves obtained using the log-rank test show C-E, the overall survival and F-H, the progression-free survival of patients with DLBCLs with high vs. low relative expression levels of miR-21, the miR-17-92 cluster, and miR-155.

Figure 2. FOXO1, which is directly targeted by miR-21 in addition to PTEN, controls Bim expression at a transcriptional level in DLBCLs

A, The expression levels of miR-21, FOXO1, and PTEN were evaluated in GCB-DLBCL and ABC-DLBCL cell lines using qRT-PCR. The bar graph shows the relative expression levels (2^−ddCt) of miR-21, FOXO1, and PTEN in each cell line compared with those of OCI-Ly1 cells. B, SU-DHL4 and SU-DHL5 cells were transfected with a miR-21 inhibitor or a negative (scramble) inhibitor using Lipofectamine 2000, and the cells were harvested at 24 hours after transfection. The expression levels of FOXO1 and PTEN in cells that were transfected with the miR-21 inhibitor were compared with those transfected with the negative inhibitor using qRT-PCR. C, OCI-Ly10 cells were transfected with miR-21 mimics or negative (scramble) mimics, and the differences in the expression levels of FOXO1 and PTEN between the negative- and miR-21 mimic-cells were assessed using qRT-PCR. D, The FOXO1 3′-untranslated region (UTR)-luciferase reporter construct containing many miR-21 binding motifs (5′-AGCUUAU-3′) was co-transfected into SU-DHL-4 and SU-DHL5 cells together with a miR-21 mimic, negative mimic, miR-21 inhibitor or negative inhibitor. At 24 hours post-transfection, the supernatant fractions of the lysed cells were recovered, and the luciferase activities were determined using a luminometer. E, At 24 hours after transfection of the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells, or miR-21 mimics or negative mimics into OCI-Ly10 cells, Western blotting was performed to determine FOXO1 level in the nucleus of tumor cells. F, Changes in the expression of FOXO1 target genes, including p27, p21, FasL and Bim, were evaluated using qRT-PCR after inhibiting the expression of miR-21 in SU-DHL4 and SU-DHL5 cells. G, A Bim promoter luciferase-reporter construct containing many copies of the FOXO1 binding motif (5′-GTAAACAA-3′) was co-transfected with either a miR-21 inhibitor or negative inhibitor into SU-DHL4 or SU-DHL5 cells. At 24 hours post-transfection, the supernatant fractions of the lysed cells were recovered, and the luciferase activities were measured using a luminometer. The values presented in the histogram are the mean values ± SD. Statistically significant differences are indicated by * and **, which signify P < 0.05 and P < 0.005, respectively, as determined using the paired t-test.

Figure 3. MiR-21 regulates the PI3K/AKT/mTOR/FOXO1 pathway and involved in the drug resistance and proliferation of DLBCL cells

At 24 hours after transfection of A, the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells or B, miR-21 mimics or negative mimics into OCI-Ly10 cells, Western blotting was performed to determine the levels of phospho-AKT, AKT, phospho-p70S6K, p70S6K, phospho-FOXO1, FOXO1 and Bim. C, SU-DHL4, SU-DHL5, and OCI-Ly10 cells were treated with increasing doses of LY294002, a PI3K inhibitor. At 24 hours after incubation, Western blotting was performed to determine the levels of FOXO1 and Bim. D, OCI-Ly10 cells were treated with increasing doses of AS1842856, a functional inhibitor of FOXO1, and 2 hours after incubation, Western blotting was performed to determine the levels of phospho-p70S6K and p70S6K. At 24 hours after transfection of E, the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells, or F, miR-21 mimics or negative mimics into OCI-Ly10 cells, the expression levels of ABCB1 (MDR1) and ABCG2 were evaluated using qRT-PCR. G, SU-DHL4 and SU-DHL5 cells were treated with the miR-21 inhibitor or negative inhibitor for 24 hours and co-incubated with the efflux-blocking drug, vinblastine, for the last 30 minutes. The cells were then stained with DiOC₂, and their drug efflux activity was analyzed. A decrease in the percentage of DiOC₂-staining cells determined using FACS represents an increase in the population of cells with drug efflux activity. H, The effect of miR-21 inhibition on the doxorubicin-induced cytotoxicity in SU-DHL4 and SU-DHL5 cells was evaluated using the CCK8 assay. I, The effect of miR-21 overexpression on the doxorubicin-induced cytotoxicity in OCI-Ly10 cells was assessed using the CCK8 assay. J, The relative rates of cell proliferation of OCI-Ly10 cells treated with DMSO (control) and doxorubicin and/or the FOXO1 inhibitor (AS1842856) were determined using the CCK8 assay. The values presented in the histogram are the mean values ± SD. Statistically significant differences are indicated by *, ** and ***, which signify P < 0.05, P < 0.005 and P < 0.0005, respectively, as determined using the paired t-test.

Figure 4. A model illustrating the regulation of the PI3K/AKT/mTOR/FOXO1 pathway by miR-21 at multiple levels in DLBCL