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

Go H, Jang JY, Kim PJ, Kim YG, Nam SJ, Paik JH, Kim TM, Heo DS, Kim CW, Jeon YK - Oncotarget (2015)

Bottom Line: 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).MiR-21 also down-regulated PTEN expression and consequently activated the PI3K/AKT/mTOR pathway, which further decreased FOXO1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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

Mentions: Given that the expression of FOXO1 and PTEN was down-regulated by miR-21 in DLBCLs (Fig. 2B and C), we further investigated if miR-21 activated the PI3K/AKT/mTOR pathway. MiR-21 inhibitor suppressed AKT and mTOR activity as shown by decrease in phospho-AKT and phospho-70S6K in SU-DHL4 and SU-DHL5 cells (Fig. 3A). At the same time, the phospho-FOXO1 level was diminished, but the amount of total FOXO1 was increased along with Bim up-regulation (Fig. 3A). In contrast, miR-21 mimics activated the PI3K/AKT/mTOR pathway in OCI-Ly10 cells but down-regulated FOXO1 and Bim (Fig. 3B). These data suggested that miR-21 activates the PI3K/AKT/mTOR pathway in DLBCL. Accordingly, miR-21 inhibition rescued FOXO1 from AKT activity-mediated phosphorylation and putatively subsequent degradation. Consistently, a functional inhibitor of PI3K, LY294002, up-regulated the expression of FOXO1 and Bim in DLBCL cells (Fig. 3C), which further suggested that the FOXO1/Bim axis may be an important target regulated by the PI3K/AKT signaling in DLBCL.


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

Go H, Jang JY, Kim PJ, Kim YG, Nam SJ, Paik JH, Kim TM, Heo DS, Kim CW, Jeon YK - Oncotarget (2015)

MiR-21 regulates the PI3K/AKT/mTOR/FOXO1 pathway and involved in the drug resistance and proliferation of DLBCL cellsAt 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.
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Figure 3: MiR-21 regulates the PI3K/AKT/mTOR/FOXO1 pathway and involved in the drug resistance and proliferation of DLBCL cellsAt 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.
Mentions: Given that the expression of FOXO1 and PTEN was down-regulated by miR-21 in DLBCLs (Fig. 2B and C), we further investigated if miR-21 activated the PI3K/AKT/mTOR pathway. MiR-21 inhibitor suppressed AKT and mTOR activity as shown by decrease in phospho-AKT and phospho-70S6K in SU-DHL4 and SU-DHL5 cells (Fig. 3A). At the same time, the phospho-FOXO1 level was diminished, but the amount of total FOXO1 was increased along with Bim up-regulation (Fig. 3A). In contrast, miR-21 mimics activated the PI3K/AKT/mTOR pathway in OCI-Ly10 cells but down-regulated FOXO1 and Bim (Fig. 3B). These data suggested that miR-21 activates the PI3K/AKT/mTOR pathway in DLBCL. Accordingly, miR-21 inhibition rescued FOXO1 from AKT activity-mediated phosphorylation and putatively subsequent degradation. Consistently, a functional inhibitor of PI3K, LY294002, up-regulated the expression of FOXO1 and Bim in DLBCL cells (Fig. 3C), which further suggested that the FOXO1/Bim axis may be an important target regulated by the PI3K/AKT signaling in DLBCL.

Bottom Line: 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).MiR-21 also down-regulated PTEN expression and consequently activated the PI3K/AKT/mTOR pathway, which further decreased FOXO1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, South Korea.

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.

No MeSH data available.


Related in: MedlinePlus