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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

ATMIN and PARP1 mRNAs are targets of miR-124.(A) Relative luciferase activity in U-2 OS cells carrying the luciferase reporter (pGL4) linked to the sense-oriented (normally orientated) 3′-UTR of putative miR-124 targets RAD17, UBE2B, PARP1, FANCF, and ATMIN or the empty reporter vector control (VC). *Significantly different (P < 0.05) from the VC group. (B) Two predicted miR-124 binding sequences in ATMIN-3′-UTR (upper panel, ATMIN-3′-UTR) were mutated (upper panel, ATMIN-3′-UTR MT1 and ATMIN-3′-UTR MT2). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the sense-oriented (normally oriented) (forward) ATMIN-3′-UTR (miR-124-F), reverse-oriented ATMIN-3′-UTR (miR-124-R), ATMIN-3′-UTR MT1 (miR-124-MT1), ATMIN-3′-UTR MT2 (miR-124-MT2), or the mutant carrying mutations at both of the two predicted sites (miR-124-MT1&MT2). (C) One predicted miR-124 binding sequence in PARP1-3′-UTR (upper panel, PARP1-3'-UTR) was mutated (upper panel, PARP1-3'-UTR MT). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the [sense-oriented (normally oriented) (forward) PARP1-3'-UTR (miR-124-F), reverse-oriented PARP1-3'-UTR (miR-124-R, or PARP1-3'-UTR MT (miR-124-MT). (D) MiR-124 regulates expression ATMIN and PARP1. Western blot of ATMIN and PARP1 in U-2 OS cells transiently transfected with the VC or miR-124-expressing vector (miR-124; upper panel), or with a 2′-O-methyl-modified antisense inhibitor of miR-124 (Anti-miR-124; lower panel) or 2′-O-methyl negative control (Anti-miR) [48]. Tubulin was included as a protein loading control, and the numbers indicate the expression relative to expression in the VC or Anti-miR control, measured by densitometry. *P < 0.05 relative to VC (A) or to miR-124-R (B and C).
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pone.0128472.g003: ATMIN and PARP1 mRNAs are targets of miR-124.(A) Relative luciferase activity in U-2 OS cells carrying the luciferase reporter (pGL4) linked to the sense-oriented (normally orientated) 3′-UTR of putative miR-124 targets RAD17, UBE2B, PARP1, FANCF, and ATMIN or the empty reporter vector control (VC). *Significantly different (P < 0.05) from the VC group. (B) Two predicted miR-124 binding sequences in ATMIN-3′-UTR (upper panel, ATMIN-3′-UTR) were mutated (upper panel, ATMIN-3′-UTR MT1 and ATMIN-3′-UTR MT2). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the sense-oriented (normally oriented) (forward) ATMIN-3′-UTR (miR-124-F), reverse-oriented ATMIN-3′-UTR (miR-124-R), ATMIN-3′-UTR MT1 (miR-124-MT1), ATMIN-3′-UTR MT2 (miR-124-MT2), or the mutant carrying mutations at both of the two predicted sites (miR-124-MT1&MT2). (C) One predicted miR-124 binding sequence in PARP1-3′-UTR (upper panel, PARP1-3'-UTR) was mutated (upper panel, PARP1-3'-UTR MT). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the [sense-oriented (normally oriented) (forward) PARP1-3'-UTR (miR-124-F), reverse-oriented PARP1-3'-UTR (miR-124-R, or PARP1-3'-UTR MT (miR-124-MT). (D) MiR-124 regulates expression ATMIN and PARP1. Western blot of ATMIN and PARP1 in U-2 OS cells transiently transfected with the VC or miR-124-expressing vector (miR-124; upper panel), or with a 2′-O-methyl-modified antisense inhibitor of miR-124 (Anti-miR-124; lower panel) or 2′-O-methyl negative control (Anti-miR) [48]. Tubulin was included as a protein loading control, and the numbers indicate the expression relative to expression in the VC or Anti-miR control, measured by densitometry. *P < 0.05 relative to VC (A) or to miR-124-R (B and C).

Mentions: The damage type–specific effect of miR-124 that we detected prompted us to propose that miR-124 may target the genes involved in specific DNA repair pathways. Because CPT, ETO, DOX, and IR specifically cause formation of DNA strand-breaks (SBs) and the sensitivity of cancer cells to anti-tumor drugs is related to the expression of DNA repair genes [21], we were particularly interested in genes involved in DNA SB repair. To investigate potential targets of miR-124 regulation, we used the microRNA target prediction bioinformatics platforms, miRanda, TargetScan, and PicTar [32, 33]. We selected those potential target genes consistently predicted by these three platforms, resulting in 427 genes that may be the target of miR-124. To focus the genes specifically having a role in DNA repair, we used the Ingenuity Pathway Analysis [34] to specify the functions of these genes, and 81 of them were involved in DNA repair. We thoroughly and individually checked these functions, and included those only playing a direct role in DNA repair, concluding the five most likely genes, Rad17, UBE2B, PARP1, FANCF, and ATMIN. These five were examined by the following reporter assay. The wild–type full-length 3′-UTR of these genes was cloned into a firefly luciferase reporter vector. U-2 OS cells were transiently transfected with these constructs and the miR-124 overexpression vector. MiR-124 overexpression significantly suppressed the luciferase activity of the constructs containing the 3′-UTR of ATMIN and PARP1 relative to the empty expression vector control (Fig 3A). We then introduced mutations in the two predicted miR-124 binding sites in ATMIN-3′-UTR and the one predicted binding site in PARP1-3′-UTR, and we examined the effects of these mutations on the response to miR-124 overexpression in the U-2 OS cell luciferase reporter assay (Fig 3B). We found that the reporter gene activity in miR-124-overexpressing cells transfected with the construct containing the reversed ATMIN-3′-UTR was significantly greater than that in the cells transfected with the wild-type forward ATMIN-3′-UTR reporter or the empty reporter plasmid. Mutation of the second, but not the first, putative miR-124 binding site in ATMIN-3′-UTR disrupted miR-124 overexpression–induced repression, indicating that miR-124 regulates ATMIN by binding to the second site (Fig 3B). Similarly, mutation of the predicted miR-124 binding site in PARP1-3′-UTR increased luciferase reporter gene activity, confirming that the 3'-UTR of PARP1 is a binding target of miR-124 (Fig 3C). To examine whether these findings held true in vivo, we verified that ATMIN and PARP1 protein levels were affected by miR-124. Protein levels in U-2 OS cells transfected with a 2′-O-methyl-modified antisense inhibitor of miR-124 or 2′-O-methyl-negative control were examined by western blotting. With increased miR-124 expression, ATMIN and PARP1 levels substantially decreased. Conversely, inhibition of endogenous miR-124 resulted in upregulation of ATMIN and PARP1 (Fig 3D).


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)

ATMIN and PARP1 mRNAs are targets of miR-124.(A) Relative luciferase activity in U-2 OS cells carrying the luciferase reporter (pGL4) linked to the sense-oriented (normally orientated) 3′-UTR of putative miR-124 targets RAD17, UBE2B, PARP1, FANCF, and ATMIN or the empty reporter vector control (VC). *Significantly different (P < 0.05) from the VC group. (B) Two predicted miR-124 binding sequences in ATMIN-3′-UTR (upper panel, ATMIN-3′-UTR) were mutated (upper panel, ATMIN-3′-UTR MT1 and ATMIN-3′-UTR MT2). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the sense-oriented (normally oriented) (forward) ATMIN-3′-UTR (miR-124-F), reverse-oriented ATMIN-3′-UTR (miR-124-R), ATMIN-3′-UTR MT1 (miR-124-MT1), ATMIN-3′-UTR MT2 (miR-124-MT2), or the mutant carrying mutations at both of the two predicted sites (miR-124-MT1&MT2). (C) One predicted miR-124 binding sequence in PARP1-3′-UTR (upper panel, PARP1-3'-UTR) was mutated (upper panel, PARP1-3'-UTR MT). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the [sense-oriented (normally oriented) (forward) PARP1-3'-UTR (miR-124-F), reverse-oriented PARP1-3'-UTR (miR-124-R, or PARP1-3'-UTR MT (miR-124-MT). (D) MiR-124 regulates expression ATMIN and PARP1. Western blot of ATMIN and PARP1 in U-2 OS cells transiently transfected with the VC or miR-124-expressing vector (miR-124; upper panel), or with a 2′-O-methyl-modified antisense inhibitor of miR-124 (Anti-miR-124; lower panel) or 2′-O-methyl negative control (Anti-miR) [48]. Tubulin was included as a protein loading control, and the numbers indicate the expression relative to expression in the VC or Anti-miR control, measured by densitometry. *P < 0.05 relative to VC (A) or to miR-124-R (B and C).
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pone.0128472.g003: ATMIN and PARP1 mRNAs are targets of miR-124.(A) Relative luciferase activity in U-2 OS cells carrying the luciferase reporter (pGL4) linked to the sense-oriented (normally orientated) 3′-UTR of putative miR-124 targets RAD17, UBE2B, PARP1, FANCF, and ATMIN or the empty reporter vector control (VC). *Significantly different (P < 0.05) from the VC group. (B) Two predicted miR-124 binding sequences in ATMIN-3′-UTR (upper panel, ATMIN-3′-UTR) were mutated (upper panel, ATMIN-3′-UTR MT1 and ATMIN-3′-UTR MT2). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the sense-oriented (normally oriented) (forward) ATMIN-3′-UTR (miR-124-F), reverse-oriented ATMIN-3′-UTR (miR-124-R), ATMIN-3′-UTR MT1 (miR-124-MT1), ATMIN-3′-UTR MT2 (miR-124-MT2), or the mutant carrying mutations at both of the two predicted sites (miR-124-MT1&MT2). (C) One predicted miR-124 binding sequence in PARP1-3′-UTR (upper panel, PARP1-3'-UTR) was mutated (upper panel, PARP1-3'-UTR MT). (Lower panel) Expression of the reporter gene in U-2 OS cells when linked to the [sense-oriented (normally oriented) (forward) PARP1-3'-UTR (miR-124-F), reverse-oriented PARP1-3'-UTR (miR-124-R, or PARP1-3'-UTR MT (miR-124-MT). (D) MiR-124 regulates expression ATMIN and PARP1. Western blot of ATMIN and PARP1 in U-2 OS cells transiently transfected with the VC or miR-124-expressing vector (miR-124; upper panel), or with a 2′-O-methyl-modified antisense inhibitor of miR-124 (Anti-miR-124; lower panel) or 2′-O-methyl negative control (Anti-miR) [48]. Tubulin was included as a protein loading control, and the numbers indicate the expression relative to expression in the VC or Anti-miR control, measured by densitometry. *P < 0.05 relative to VC (A) or to miR-124-R (B and C).
Mentions: The damage type–specific effect of miR-124 that we detected prompted us to propose that miR-124 may target the genes involved in specific DNA repair pathways. Because CPT, ETO, DOX, and IR specifically cause formation of DNA strand-breaks (SBs) and the sensitivity of cancer cells to anti-tumor drugs is related to the expression of DNA repair genes [21], we were particularly interested in genes involved in DNA SB repair. To investigate potential targets of miR-124 regulation, we used the microRNA target prediction bioinformatics platforms, miRanda, TargetScan, and PicTar [32, 33]. We selected those potential target genes consistently predicted by these three platforms, resulting in 427 genes that may be the target of miR-124. To focus the genes specifically having a role in DNA repair, we used the Ingenuity Pathway Analysis [34] to specify the functions of these genes, and 81 of them were involved in DNA repair. We thoroughly and individually checked these functions, and included those only playing a direct role in DNA repair, concluding the five most likely genes, Rad17, UBE2B, PARP1, FANCF, and ATMIN. These five were examined by the following reporter assay. The wild–type full-length 3′-UTR of these genes was cloned into a firefly luciferase reporter vector. U-2 OS cells were transiently transfected with these constructs and the miR-124 overexpression vector. MiR-124 overexpression significantly suppressed the luciferase activity of the constructs containing the 3′-UTR of ATMIN and PARP1 relative to the empty expression vector control (Fig 3A). We then introduced mutations in the two predicted miR-124 binding sites in ATMIN-3′-UTR and the one predicted binding site in PARP1-3′-UTR, and we examined the effects of these mutations on the response to miR-124 overexpression in the U-2 OS cell luciferase reporter assay (Fig 3B). We found that the reporter gene activity in miR-124-overexpressing cells transfected with the construct containing the reversed ATMIN-3′-UTR was significantly greater than that in the cells transfected with the wild-type forward ATMIN-3′-UTR reporter or the empty reporter plasmid. Mutation of the second, but not the first, putative miR-124 binding site in ATMIN-3′-UTR disrupted miR-124 overexpression–induced repression, indicating that miR-124 regulates ATMIN by binding to the second site (Fig 3B). Similarly, mutation of the predicted miR-124 binding site in PARP1-3′-UTR increased luciferase reporter gene activity, confirming that the 3'-UTR of PARP1 is a binding target of miR-124 (Fig 3C). To examine whether these findings held true in vivo, we verified that ATMIN and PARP1 protein levels were affected by miR-124. Protein levels in U-2 OS cells transfected with a 2′-O-methyl-modified antisense inhibitor of miR-124 or 2′-O-methyl-negative control were examined by western blotting. With increased miR-124 expression, ATMIN and PARP1 levels substantially decreased. Conversely, inhibition of endogenous miR-124 resulted in upregulation of ATMIN and PARP1 (Fig 3D).

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