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miR-17* suppresses tumorigenicity of prostate cancer by inhibiting mitochondrial antioxidant enzymes.

Xu Y, Fang F, Zhang J, Josson S, St Clair WH, St Clair DK - PLoS ONE (2010)

Bottom Line: Transfection of miR-17* into prostate cancer PC-3 cells significantly reduces levels of the three antioxidant proteins and activity of the luciferase reporter under the control of miR-17* binding sequences located in the 3'-untranslated regions of the three target genes.Disulfiram (DSF), a dithiolcarbomate drug shown to have an anticancer effect, induces the level of mature miR-17* and cell death in PCa cells, which can be attenuated by transfection of antisense miR-17*.These results suggest that miR-17* may suppress tumorigenicity of prostate cancer through inhibition of mitochondrial antioxidant enzymes.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Aberrant micro RNA (miRNA) expression has been implicated in the pathogenesis of cancer. Recent studies have shown that the miR-17-92 cluster is overexpressed in many types of cancer. The oncogenic function of mature miRNAs encoded by the miR-17-92 cluster has been identified from the 5' arm of six precursors. However, the function of the miRNAs produced from the 3' arm of these precursors remains unknown. The present study demonstrates that miR-17* is able to suppress critical primary mitochondrial antioxidant enzymes, such as manganese superoxide dismutase (MnSOD), glutathione peroxidase-2 (GPX2) and thioredoxin reductase-2 (TrxR2). Transfection of miR-17* into prostate cancer PC-3 cells significantly reduces levels of the three antioxidant proteins and activity of the luciferase reporter under the control of miR-17* binding sequences located in the 3'-untranslated regions of the three target genes. Disulfiram (DSF), a dithiolcarbomate drug shown to have an anticancer effect, induces the level of mature miR-17* and cell death in PCa cells, which can be attenuated by transfection of antisense miR-17*. Increasing miR-17* level in PC-3 cells by a Tet-on based conditional expression system markedly suppresses its tumorigencity. These results suggest that miR-17* may suppress tumorigenicity of prostate cancer through inhibition of mitochondrial antioxidant enzymes.

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Induction of miR-17* in PCa cells by DSF.A, PCa cells were treated with DSF at indicated concentrations. The levels of the three antioxidant proteins were measured by Western blots. B, mRNA levels of the three antioxidant genes were quantified by RT-PCR. C, the levels of miR-17 and miR-17* in the DSF-treated cells were quantified by RT-PCR. The miR-17* levels were confirmed by Northern blots. D, the effect of DSF-induced miR-17* on the reporter responses was determined. E, after transfected anti-miR-17*, PC-3 cells were treated with DSF. The effect of anti-miR-17* on restoring antioxidant proteins was quantified by Western blots. β-actin was used to normalize the levels of proteins (A), (E), and the levels of mRNA (B). The fold changes are indicated. RNU24 was used to normalize the levels of miR-17 and miR-17* (C). β-gal activity was used to normalize luciferase reporter activities (D). Three samples (n = 3) were used in the experiments (with the exception of Northern blot). * (p<0.05) and ** (p<0.01) indicate significances as compared to no DSF treatment (A), (C), (D), and compared to no DSF and no miRNA transfected samples (E).
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pone-0014356-g002: Induction of miR-17* in PCa cells by DSF.A, PCa cells were treated with DSF at indicated concentrations. The levels of the three antioxidant proteins were measured by Western blots. B, mRNA levels of the three antioxidant genes were quantified by RT-PCR. C, the levels of miR-17 and miR-17* in the DSF-treated cells were quantified by RT-PCR. The miR-17* levels were confirmed by Northern blots. D, the effect of DSF-induced miR-17* on the reporter responses was determined. E, after transfected anti-miR-17*, PC-3 cells were treated with DSF. The effect of anti-miR-17* on restoring antioxidant proteins was quantified by Western blots. β-actin was used to normalize the levels of proteins (A), (E), and the levels of mRNA (B). The fold changes are indicated. RNU24 was used to normalize the levels of miR-17 and miR-17* (C). β-gal activity was used to normalize luciferase reporter activities (D). Three samples (n = 3) were used in the experiments (with the exception of Northern blot). * (p<0.05) and ** (p<0.01) indicate significances as compared to no DSF treatment (A), (C), (D), and compared to no DSF and no miRNA transfected samples (E).

Mentions: DSF is a dithiolcarbomate drug that has been shown to suppress cancerous phenotypes by inducing the apoptotic pathway [20]. We found that DSF inhibits the three antioxidant proteins in PCa cells. After PC-3 and DU-145 cells were treated with DSF for 24 h, the reduction of the levels of the antioxidant proteins corresponded significantly with the concentrations of DSF (Fig. 2A). However, the mRNA levels of the antioxidant genes were not changed in DSF-treated cells (Fig. 2B). Interestingly, RT-PCR and Northern blots show that DSF induces miR-17* but has no effect on expression levels of miR-17 (Fig. 2C). The induction of miR-17* by DSF is further confirmed by reporter responses that are regulated by miR-17* targeting sequences (Fig. 2D). Furthermore, to verify that the negative effect of DSF on the expression of antioxidant proteins is mediated by the induction of miR-17*, the PC-3 cells were transfected with antisense hsa-miR-17* followed by DSF treatment. The results indicate that the antisense hsa-miR-17* is able to reduce the DSF effect (Fig. 2E). These results suggest that the reduction of antioxidant proteins by DSF occurs, at least in part, through miR-17*-mediated translational repression.


miR-17* suppresses tumorigenicity of prostate cancer by inhibiting mitochondrial antioxidant enzymes.

Xu Y, Fang F, Zhang J, Josson S, St Clair WH, St Clair DK - PLoS ONE (2010)

Induction of miR-17* in PCa cells by DSF.A, PCa cells were treated with DSF at indicated concentrations. The levels of the three antioxidant proteins were measured by Western blots. B, mRNA levels of the three antioxidant genes were quantified by RT-PCR. C, the levels of miR-17 and miR-17* in the DSF-treated cells were quantified by RT-PCR. The miR-17* levels were confirmed by Northern blots. D, the effect of DSF-induced miR-17* on the reporter responses was determined. E, after transfected anti-miR-17*, PC-3 cells were treated with DSF. The effect of anti-miR-17* on restoring antioxidant proteins was quantified by Western blots. β-actin was used to normalize the levels of proteins (A), (E), and the levels of mRNA (B). The fold changes are indicated. RNU24 was used to normalize the levels of miR-17 and miR-17* (C). β-gal activity was used to normalize luciferase reporter activities (D). Three samples (n = 3) were used in the experiments (with the exception of Northern blot). * (p<0.05) and ** (p<0.01) indicate significances as compared to no DSF treatment (A), (C), (D), and compared to no DSF and no miRNA transfected samples (E).
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pone-0014356-g002: Induction of miR-17* in PCa cells by DSF.A, PCa cells were treated with DSF at indicated concentrations. The levels of the three antioxidant proteins were measured by Western blots. B, mRNA levels of the three antioxidant genes were quantified by RT-PCR. C, the levels of miR-17 and miR-17* in the DSF-treated cells were quantified by RT-PCR. The miR-17* levels were confirmed by Northern blots. D, the effect of DSF-induced miR-17* on the reporter responses was determined. E, after transfected anti-miR-17*, PC-3 cells were treated with DSF. The effect of anti-miR-17* on restoring antioxidant proteins was quantified by Western blots. β-actin was used to normalize the levels of proteins (A), (E), and the levels of mRNA (B). The fold changes are indicated. RNU24 was used to normalize the levels of miR-17 and miR-17* (C). β-gal activity was used to normalize luciferase reporter activities (D). Three samples (n = 3) were used in the experiments (with the exception of Northern blot). * (p<0.05) and ** (p<0.01) indicate significances as compared to no DSF treatment (A), (C), (D), and compared to no DSF and no miRNA transfected samples (E).
Mentions: DSF is a dithiolcarbomate drug that has been shown to suppress cancerous phenotypes by inducing the apoptotic pathway [20]. We found that DSF inhibits the three antioxidant proteins in PCa cells. After PC-3 and DU-145 cells were treated with DSF for 24 h, the reduction of the levels of the antioxidant proteins corresponded significantly with the concentrations of DSF (Fig. 2A). However, the mRNA levels of the antioxidant genes were not changed in DSF-treated cells (Fig. 2B). Interestingly, RT-PCR and Northern blots show that DSF induces miR-17* but has no effect on expression levels of miR-17 (Fig. 2C). The induction of miR-17* by DSF is further confirmed by reporter responses that are regulated by miR-17* targeting sequences (Fig. 2D). Furthermore, to verify that the negative effect of DSF on the expression of antioxidant proteins is mediated by the induction of miR-17*, the PC-3 cells were transfected with antisense hsa-miR-17* followed by DSF treatment. The results indicate that the antisense hsa-miR-17* is able to reduce the DSF effect (Fig. 2E). These results suggest that the reduction of antioxidant proteins by DSF occurs, at least in part, through miR-17*-mediated translational repression.

Bottom Line: Transfection of miR-17* into prostate cancer PC-3 cells significantly reduces levels of the three antioxidant proteins and activity of the luciferase reporter under the control of miR-17* binding sequences located in the 3'-untranslated regions of the three target genes.Disulfiram (DSF), a dithiolcarbomate drug shown to have an anticancer effect, induces the level of mature miR-17* and cell death in PCa cells, which can be attenuated by transfection of antisense miR-17*.These results suggest that miR-17* may suppress tumorigenicity of prostate cancer through inhibition of mitochondrial antioxidant enzymes.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Aberrant micro RNA (miRNA) expression has been implicated in the pathogenesis of cancer. Recent studies have shown that the miR-17-92 cluster is overexpressed in many types of cancer. The oncogenic function of mature miRNAs encoded by the miR-17-92 cluster has been identified from the 5' arm of six precursors. However, the function of the miRNAs produced from the 3' arm of these precursors remains unknown. The present study demonstrates that miR-17* is able to suppress critical primary mitochondrial antioxidant enzymes, such as manganese superoxide dismutase (MnSOD), glutathione peroxidase-2 (GPX2) and thioredoxin reductase-2 (TrxR2). Transfection of miR-17* into prostate cancer PC-3 cells significantly reduces levels of the three antioxidant proteins and activity of the luciferase reporter under the control of miR-17* binding sequences located in the 3'-untranslated regions of the three target genes. Disulfiram (DSF), a dithiolcarbomate drug shown to have an anticancer effect, induces the level of mature miR-17* and cell death in PCa cells, which can be attenuated by transfection of antisense miR-17*. Increasing miR-17* level in PC-3 cells by a Tet-on based conditional expression system markedly suppresses its tumorigencity. These results suggest that miR-17* may suppress tumorigenicity of prostate cancer through inhibition of mitochondrial antioxidant enzymes.

Show MeSH
Related in: MedlinePlus