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The Effect of MicroRNA-124 Overexpression on Anti-Tumor Drug Sensitivity.

Chen SM, Chou WC, Hu LY, Hsiung CN, Chu HW, Huang YL, Hsu HM, Yu JC, Shen CY - PLoS ONE (2015)

Bottom Line: In the present study, we found that overexpression of miR-124 was associated with reduced DNA repair capacity in cultured cancer cells and increased sensitivity of cells to DNA-damaging anti-tumor drugs, specifically those that cause the formation of DNA strand-breaks (SBs).The translational relevance of this role of miR-124 in anti-tumor drug sensitivity is suggested by the finding that increased miR-124 expression correlates with better breast cancer prognosis, specifically in patients receiving chemotherapy.These findings suggest that miR-124 could potentially be used as a therapeutic agent to improve the efficacy of chemotherapy with DNA-damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

ABSTRACT
MicroRNAs play critical roles in regulating various physiological processes, including growth and development. Previous studies have shown that microRNA-124 (miR-124) participates not only in regulation of early neurogenesis but also in suppression of tumorigenesis. In the present study, we found that overexpression of miR-124 was associated with reduced DNA repair capacity in cultured cancer cells and increased sensitivity of cells to DNA-damaging anti-tumor drugs, specifically those that cause the formation of DNA strand-breaks (SBs). We then examined which DNA repair-related genes, particularly the genes of SB repair, were regulated by miR-124. Two SB repair-related genes, encoding ATM interactor (ATMIN) and poly (ADP-ribose) polymerase 1 (PARP1), were strongly affected by miR-124 overexpression, by binding of miR-124 to the 3¢-untranslated region of their mRNAs. As a result, the capacity of cells to repair DNA SBs, such as those resulting from homologous recombination, was significantly reduced upon miR-124 overexpression. A particularly important therapeutic implication of this finding is that overexpression of miR-124 enhanced cell sensitivity to multiple DNA-damaging agents via ATMIN- and PARP1-mediated mechanisms. The translational relevance of this role of miR-124 in anti-tumor drug sensitivity is suggested by the finding that increased miR-124 expression correlates with better breast cancer prognosis, specifically in patients receiving chemotherapy. These findings suggest that miR-124 could potentially be used as a therapeutic agent to improve the efficacy of chemotherapy with DNA-damaging agents.

No MeSH data available.


Related in: MedlinePlus

MiR-124 overexpression reduces DNA SB repair capacity.(A) HR repair capacity in miR-124-overexpressing cells and cells transfected with the antisense microRNA control (anti-miR-CON) or antisense miR-124 inhibitor (anti-miR-124). (B) Comet assay showing that miR-124 overexpression (miR-124) reduces SB repair relative to the vector control (VC) after treatment with CPT. (C) Immunostaining of DNA SBs (detected by the presence of γ-H2AX) in cells after IR treatment. Red: anti-γ-H2AX; blue: nuclei (left panel). The γ-H2AX signal was quantified and compared with the cells transfected with VC or miR-124 (right panel). All of the experiments shown were conducted in U-2 OS cells, and similar results were obtained in MDA-MB-231 cells. *P < 0.05 in individual comparisons.
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pone.0128472.g004: MiR-124 overexpression reduces DNA SB repair capacity.(A) HR repair capacity in miR-124-overexpressing cells and cells transfected with the antisense microRNA control (anti-miR-CON) or antisense miR-124 inhibitor (anti-miR-124). (B) Comet assay showing that miR-124 overexpression (miR-124) reduces SB repair relative to the vector control (VC) after treatment with CPT. (C) Immunostaining of DNA SBs (detected by the presence of γ-H2AX) in cells after IR treatment. Red: anti-γ-H2AX; blue: nuclei (left panel). The γ-H2AX signal was quantified and compared with the cells transfected with VC or miR-124 (right panel). All of the experiments shown were conducted in U-2 OS cells, and similar results were obtained in MDA-MB-231 cells. *P < 0.05 in individual comparisons.

Mentions: The identification of ATMIN and PARP1 as potential targets of miR-124 prompted us to further test the effects of miR-124 on DNA SB repair. We thus examined homologous recombination (HR) repair—the pathway particularly important in repairing SBs—in cells using an established system based on strand exchange between two differently mutated enhanced green fluorescent protein gene sequences [35]. In this system, miR-124 overexpression reduced HR repair capacity to 40% of the vector control, whereas inhibition of endogenous miR-124 had no significant effect on HR repair (Fig 4A). We next performed a single-cell gel electrophoresis (comet) assay to further examine the extent of DNA damage/repair in cells overexpressing miR-124 after treatment with CPT for 1 h (S3 Fig). Cells overexpressing miR-124 had significantly different comet profiles than the vector control group, indicating that more DNA SB damage remained and, hence, a lower repair capacity, especially in the 3 h recovery from CPT group (Fig 4B). Because histone H2AX phosphorylation is a well-known marker of DNA SBs, we used immunostaining of phosphorylated H2AX (γ-H2AX) to examine the distribution of DNA damage after IR treatment. The miR-124-overexpressing and the vector control groups had similar proportions of γ-H2AX-positive cells after 30 min recovery from IR, but the miR-124-overexpressing cells appeared to have more γ-H2AX foci per cell than the vector control cells, suggesting more DNA damage in the miR-124-overexpressing cells (Fig 4C).


The Effect of MicroRNA-124 Overexpression on Anti-Tumor Drug Sensitivity.

Chen SM, Chou WC, Hu LY, Hsiung CN, Chu HW, Huang YL, Hsu HM, Yu JC, Shen CY - PLoS ONE (2015)

MiR-124 overexpression reduces DNA SB repair capacity.(A) HR repair capacity in miR-124-overexpressing cells and cells transfected with the antisense microRNA control (anti-miR-CON) or antisense miR-124 inhibitor (anti-miR-124). (B) Comet assay showing that miR-124 overexpression (miR-124) reduces SB repair relative to the vector control (VC) after treatment with CPT. (C) Immunostaining of DNA SBs (detected by the presence of γ-H2AX) in cells after IR treatment. Red: anti-γ-H2AX; blue: nuclei (left panel). The γ-H2AX signal was quantified and compared with the cells transfected with VC or miR-124 (right panel). All of the experiments shown were conducted in U-2 OS cells, and similar results were obtained in MDA-MB-231 cells. *P < 0.05 in individual comparisons.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482746&req=5

pone.0128472.g004: MiR-124 overexpression reduces DNA SB repair capacity.(A) HR repair capacity in miR-124-overexpressing cells and cells transfected with the antisense microRNA control (anti-miR-CON) or antisense miR-124 inhibitor (anti-miR-124). (B) Comet assay showing that miR-124 overexpression (miR-124) reduces SB repair relative to the vector control (VC) after treatment with CPT. (C) Immunostaining of DNA SBs (detected by the presence of γ-H2AX) in cells after IR treatment. Red: anti-γ-H2AX; blue: nuclei (left panel). The γ-H2AX signal was quantified and compared with the cells transfected with VC or miR-124 (right panel). All of the experiments shown were conducted in U-2 OS cells, and similar results were obtained in MDA-MB-231 cells. *P < 0.05 in individual comparisons.
Mentions: The identification of ATMIN and PARP1 as potential targets of miR-124 prompted us to further test the effects of miR-124 on DNA SB repair. We thus examined homologous recombination (HR) repair—the pathway particularly important in repairing SBs—in cells using an established system based on strand exchange between two differently mutated enhanced green fluorescent protein gene sequences [35]. In this system, miR-124 overexpression reduced HR repair capacity to 40% of the vector control, whereas inhibition of endogenous miR-124 had no significant effect on HR repair (Fig 4A). We next performed a single-cell gel electrophoresis (comet) assay to further examine the extent of DNA damage/repair in cells overexpressing miR-124 after treatment with CPT for 1 h (S3 Fig). Cells overexpressing miR-124 had significantly different comet profiles than the vector control group, indicating that more DNA SB damage remained and, hence, a lower repair capacity, especially in the 3 h recovery from CPT group (Fig 4B). Because histone H2AX phosphorylation is a well-known marker of DNA SBs, we used immunostaining of phosphorylated H2AX (γ-H2AX) to examine the distribution of DNA damage after IR treatment. The miR-124-overexpressing and the vector control groups had similar proportions of γ-H2AX-positive cells after 30 min recovery from IR, but the miR-124-overexpressing cells appeared to have more γ-H2AX foci per cell than the vector control cells, suggesting more DNA damage in the miR-124-overexpressing cells (Fig 4C).

Bottom Line: In the present study, we found that overexpression of miR-124 was associated with reduced DNA repair capacity in cultured cancer cells and increased sensitivity of cells to DNA-damaging anti-tumor drugs, specifically those that cause the formation of DNA strand-breaks (SBs).The translational relevance of this role of miR-124 in anti-tumor drug sensitivity is suggested by the finding that increased miR-124 expression correlates with better breast cancer prognosis, specifically in patients receiving chemotherapy.These findings suggest that miR-124 could potentially be used as a therapeutic agent to improve the efficacy of chemotherapy with DNA-damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

ABSTRACT
MicroRNAs play critical roles in regulating various physiological processes, including growth and development. Previous studies have shown that microRNA-124 (miR-124) participates not only in regulation of early neurogenesis but also in suppression of tumorigenesis. In the present study, we found that overexpression of miR-124 was associated with reduced DNA repair capacity in cultured cancer cells and increased sensitivity of cells to DNA-damaging anti-tumor drugs, specifically those that cause the formation of DNA strand-breaks (SBs). We then examined which DNA repair-related genes, particularly the genes of SB repair, were regulated by miR-124. Two SB repair-related genes, encoding ATM interactor (ATMIN) and poly (ADP-ribose) polymerase 1 (PARP1), were strongly affected by miR-124 overexpression, by binding of miR-124 to the 3¢-untranslated region of their mRNAs. As a result, the capacity of cells to repair DNA SBs, such as those resulting from homologous recombination, was significantly reduced upon miR-124 overexpression. A particularly important therapeutic implication of this finding is that overexpression of miR-124 enhanced cell sensitivity to multiple DNA-damaging agents via ATMIN- and PARP1-mediated mechanisms. The translational relevance of this role of miR-124 in anti-tumor drug sensitivity is suggested by the finding that increased miR-124 expression correlates with better breast cancer prognosis, specifically in patients receiving chemotherapy. These findings suggest that miR-124 could potentially be used as a therapeutic agent to improve the efficacy of chemotherapy with DNA-damaging agents.

No MeSH data available.


Related in: MedlinePlus