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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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Cytotoxicity of miR-17* in PCa cells.A, the PCa cells were treated with DSF at the indicated concentrations for colony survival analysis. The formed colonies were counted and plotted in a log scale. B, the PC-3 cells were transfected with miR-17* and control miRNAs prior to the DSF treatment. The effects of miR-17* and antisense miR-17* on colony survival were determined. C and D, miR-17* was co-transfected with constructs for expression of the three antioxidant proteins. The overexpressed antioxidant proteins were confirmed by Western blots with β-actin normalization and fold changes are indicated (D). Protective effects of the transfected antioxidant enzymes on the cells against miR-17* toxicity were determined by a trypan blue exclusion assay (C). Three samples (n = 3) were used in the experiments. * (p<0.05) and ** (p<0.01) indicate significances as compared to control miRNA samples (B) and compared to vehicle control samples (C), (D).
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pone-0014356-g003: Cytotoxicity of miR-17* in PCa cells.A, the PCa cells were treated with DSF at the indicated concentrations for colony survival analysis. The formed colonies were counted and plotted in a log scale. B, the PC-3 cells were transfected with miR-17* and control miRNAs prior to the DSF treatment. The effects of miR-17* and antisense miR-17* on colony survival were determined. C and D, miR-17* was co-transfected with constructs for expression of the three antioxidant proteins. The overexpressed antioxidant proteins were confirmed by Western blots with β-actin normalization and fold changes are indicated (D). Protective effects of the transfected antioxidant enzymes on the cells against miR-17* toxicity were determined by a trypan blue exclusion assay (C). Three samples (n = 3) were used in the experiments. * (p<0.05) and ** (p<0.01) indicate significances as compared to control miRNA samples (B) and compared to vehicle control samples (C), (D).

Mentions: To determine DSF toxicity to PCa cells, PC-3 and DU-145 cells were treated with DSF and cultured until colonies formed. Because the cell density used for colony formation analysis was 100-fold less than the density shown in Fig. 2, the concentration range of DSF was reduced 100-fold in colony formation experiments. The results of survival fraction indicate that PCa cells are extremely sensitive to DSF. More than 95% of the cells were killed by treatment with 1 µM DSF (Fig. 3A). To verify whether miR-17* contributes to DSF-mediated cell death, PC-3 cells were transfected with miR-17* and anti-miR-17* prior to DSF treatment. Colony survival analysis shows that miR-17* enhances the toxicity of DSF, whereas anti-miR-17* is able to rescue cells from the DSF effect (Fig. 3B). To further verify that reduction of antioxidant proteins is a major cause for the toxicity of miR-17*, the PC-3 cells were co-transfected with miR-17* and cDNAs ectopically expressing the three antioxidant proteins that are not susceptible to miR-17* regulation. Cytotoxicity analysis by trypan blue exclusion assay shows that expression of the three antioxidant genes rescues cell survival from the toxicity of miR-17* (Fig. 3C). The corresponding levels of the antioxidant proteins in the transfected PC-3 cells were verified by Western blots (Fig. 3D).


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)

Cytotoxicity of miR-17* in PCa cells.A, the PCa cells were treated with DSF at the indicated concentrations for colony survival analysis. The formed colonies were counted and plotted in a log scale. B, the PC-3 cells were transfected with miR-17* and control miRNAs prior to the DSF treatment. The effects of miR-17* and antisense miR-17* on colony survival were determined. C and D, miR-17* was co-transfected with constructs for expression of the three antioxidant proteins. The overexpressed antioxidant proteins were confirmed by Western blots with β-actin normalization and fold changes are indicated (D). Protective effects of the transfected antioxidant enzymes on the cells against miR-17* toxicity were determined by a trypan blue exclusion assay (C). Three samples (n = 3) were used in the experiments. * (p<0.05) and ** (p<0.01) indicate significances as compared to control miRNA samples (B) and compared to vehicle control samples (C), (D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008681&req=5

pone-0014356-g003: Cytotoxicity of miR-17* in PCa cells.A, the PCa cells were treated with DSF at the indicated concentrations for colony survival analysis. The formed colonies were counted and plotted in a log scale. B, the PC-3 cells were transfected with miR-17* and control miRNAs prior to the DSF treatment. The effects of miR-17* and antisense miR-17* on colony survival were determined. C and D, miR-17* was co-transfected with constructs for expression of the three antioxidant proteins. The overexpressed antioxidant proteins were confirmed by Western blots with β-actin normalization and fold changes are indicated (D). Protective effects of the transfected antioxidant enzymes on the cells against miR-17* toxicity were determined by a trypan blue exclusion assay (C). Three samples (n = 3) were used in the experiments. * (p<0.05) and ** (p<0.01) indicate significances as compared to control miRNA samples (B) and compared to vehicle control samples (C), (D).
Mentions: To determine DSF toxicity to PCa cells, PC-3 and DU-145 cells were treated with DSF and cultured until colonies formed. Because the cell density used for colony formation analysis was 100-fold less than the density shown in Fig. 2, the concentration range of DSF was reduced 100-fold in colony formation experiments. The results of survival fraction indicate that PCa cells are extremely sensitive to DSF. More than 95% of the cells were killed by treatment with 1 µM DSF (Fig. 3A). To verify whether miR-17* contributes to DSF-mediated cell death, PC-3 cells were transfected with miR-17* and anti-miR-17* prior to DSF treatment. Colony survival analysis shows that miR-17* enhances the toxicity of DSF, whereas anti-miR-17* is able to rescue cells from the DSF effect (Fig. 3B). To further verify that reduction of antioxidant proteins is a major cause for the toxicity of miR-17*, the PC-3 cells were co-transfected with miR-17* and cDNAs ectopically expressing the three antioxidant proteins that are not susceptible to miR-17* regulation. Cytotoxicity analysis by trypan blue exclusion assay shows that expression of the three antioxidant genes rescues cell survival from the toxicity of miR-17* (Fig. 3C). The corresponding levels of the antioxidant proteins in the transfected PC-3 cells were verified by Western blots (Fig. 3D).

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