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MiR-630 inhibits proliferation by targeting CDC7 kinase, but maintains the apoptotic balance by targeting multiple modulators in human lung cancer A549 cells.

Cao JX, Lu Y, Qi JJ, An GS, Mao ZB, Jia HT, Li SY, Ni JH - Cell Death Dis (2014)

Bottom Line: We found that miR-630 was highly expressed in A549 and NIH3T3 cells where CDC7 was downregulated, but lower in H1299, MCF7, MDA-MB-231, HeLa and 2BS cells where CDC7 was upregulated.Our data also support the notion that a certain mRNA can be targeted by several miRNAs, and in particular an miRNA may target a set of mRNAs.These data afford a comprehensive view of microRNA-dependent control of gene expression in the regulation of apoptosis under genotoxic stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Xue Yuan Road 38, Beijing 100191, People's Republic of China.

ABSTRACT
MicroRNAome analyses have shown microRNA-630 (miR-630) to be involved in the regulation of apoptosis. However, its apoptotic role is still debated and its participation in DNA replication is unknown. Here, we demonstrate that miR-630 inhibits cell proliferation by targeting cell-cycle kinase 7 (CDC7) kinase, but maintains the apoptotic balance by targeting multiple activators of apoptosis under genotoxic stress. We identified a novel regulatory mechanism of CDC7 gene expression, in which miR-630 downregulated CDC7 expression by recognizing and binding to four binding sites in CDC7 3'-UTR. We found that miR-630 was highly expressed in A549 and NIH3T3 cells where CDC7 was downregulated, but lower in H1299, MCF7, MDA-MB-231, HeLa and 2BS cells where CDC7 was upregulated. Furthermore, the induction of miR-630 occurred commonly in a variety of human cancer and immortalized cells in response to genotoxic agents. Importantly, downregulation of CDC7 by miR-630 was associated with cisplatin (CIS)-induced inhibitory proliferation in A549 cells. Mechanistically, miR-630 exerted its inhibitory proliferation by blocking CDC7-mediated initiation of DNA synthesis and by inducing G1 arrest, but maintains apoptotic balance under CIS exposure. On the one hand, miR-630 promoted apoptosis by downregulation of CDC7; on the other hand, it reduced apoptosis by downregulating several apoptotic modulators such as PARP3, DDIT4, EP300 and EP300 downstream effector p53, thereby maintaining the apoptotic balance. Our data indicate that miR-630 has a bimodal role in the regulation of apoptosis in response to DNA damage. Our data also support the notion that a certain mRNA can be targeted by several miRNAs, and in particular an miRNA may target a set of mRNAs. These data afford a comprehensive view of microRNA-dependent control of gene expression in the regulation of apoptosis under genotoxic stress.

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Schematic diagram showing multiple target roles of miR-630 in regulating apoptosis under DNA damage stress. MiR-630 promotes apoptosis by suppressing CDC7 expression and reduces apoptosis by direct and indirect suppressing apoptotic regulators DDIT4, PARP3, EP300 and p53
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fig7: Schematic diagram showing multiple target roles of miR-630 in regulating apoptosis under DNA damage stress. MiR-630 promotes apoptosis by suppressing CDC7 expression and reduces apoptosis by direct and indirect suppressing apoptotic regulators DDIT4, PARP3, EP300 and p53

Mentions: We found that silencing CDC7 caused apoptosis in A549 (Figures 3e and a, lower panel), as CIS did so (Figures 3a–c), whereas CDC7 overexpression diminished CIS-induced apoptosis (Figures 3h and i and Supplementary Figure S5), indicating that CDC7 downregulation is responsible at least partly for CIS-induced apoptosis. Surprisingly, we could not detect apoptosis in miR-630 transfected cells (Figure 5a and Supplementary Figure S6), indicating that overexpression of miR-630 and depletion of CDC7 by RNA interference (RNAi) (or downregulation of CDC7 by CIS) do not have equal effect on apoptosis, probably because of the multiple target effects of miR-630. We showed that DDIT4, PARP3 and EP300, which have been shown to be apoptotic activators,34,38, 39, 40 were specific targets for miR-630 (Figure 5c). MiR-630 transfection significantly reduced expression of DDIT4, PARP3 and EP300 mRNAs and proteins in A549 (Figures 5d and e). Importantly, individual silencing, in particular, combined silencing of DDIT4, PARP3 and EP300, significantly inhibited apoptosis (Figures 5f and g and Supplementary Figure S8) and procaspase-3 activation (Figure 5h). Furthermore, miR-630 overexpression markedly decreased acetylated and phosphorylated p53 and total p53 in CIS-exposed A549 (Figure 5i). EP300 may act as a regulator of p53 via its acetylase.39,40 Therefore, decreased acetylation of p53 should be attributed to miR-630-targeted EP300. Our data suggest that miR-630 may directly or indirectly downregulate both apoptotic activator(s) and inhibitor(s) that have a bimodal role in the regulation of apoptosis. On the one hand, miR-630 promoted apoptosis by downregulating CDC7; on the other hand, it reduced apoptosis by downregulating apoptotic activators DDIT4, PARP3, EP300 and p53, thereby maintaining apoptotic balance in DDR (Figure 7). This can explain why we could not detect apoptosis in miR-630-transfected cells (Figure 5a). Although miR-630 transfection or induction may partly offset p53 acetylation and phosphorylation, the levels of chemomodified p53 in CIS-exposed cells were still higher than control (Figure 5i). We found recently that p53 was accumulated in HCT116p53+/+ and A549 upon CIS-induced DNA damage.54 Our recent observation54 and the data fromFigure 5i suggest that apoptotic mechanism still works in the presence of CIS, although miR-630 downregulates p53 by targeting EP300. Notably, miR-630 overexpression is an extreme case that differs from miR-630 induction by CIS. Therefore, the bimodal role of miR-630 does not discriminate against the contributions of CDC7 inactivation and other apoptotic regulators to CIS-induced apoptosis.


MiR-630 inhibits proliferation by targeting CDC7 kinase, but maintains the apoptotic balance by targeting multiple modulators in human lung cancer A549 cells.

Cao JX, Lu Y, Qi JJ, An GS, Mao ZB, Jia HT, Li SY, Ni JH - Cell Death Dis (2014)

Schematic diagram showing multiple target roles of miR-630 in regulating apoptosis under DNA damage stress. MiR-630 promotes apoptosis by suppressing CDC7 expression and reduces apoptosis by direct and indirect suppressing apoptotic regulators DDIT4, PARP3, EP300 and p53
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4225225&req=5

fig7: Schematic diagram showing multiple target roles of miR-630 in regulating apoptosis under DNA damage stress. MiR-630 promotes apoptosis by suppressing CDC7 expression and reduces apoptosis by direct and indirect suppressing apoptotic regulators DDIT4, PARP3, EP300 and p53
Mentions: We found that silencing CDC7 caused apoptosis in A549 (Figures 3e and a, lower panel), as CIS did so (Figures 3a–c), whereas CDC7 overexpression diminished CIS-induced apoptosis (Figures 3h and i and Supplementary Figure S5), indicating that CDC7 downregulation is responsible at least partly for CIS-induced apoptosis. Surprisingly, we could not detect apoptosis in miR-630 transfected cells (Figure 5a and Supplementary Figure S6), indicating that overexpression of miR-630 and depletion of CDC7 by RNA interference (RNAi) (or downregulation of CDC7 by CIS) do not have equal effect on apoptosis, probably because of the multiple target effects of miR-630. We showed that DDIT4, PARP3 and EP300, which have been shown to be apoptotic activators,34,38, 39, 40 were specific targets for miR-630 (Figure 5c). MiR-630 transfection significantly reduced expression of DDIT4, PARP3 and EP300 mRNAs and proteins in A549 (Figures 5d and e). Importantly, individual silencing, in particular, combined silencing of DDIT4, PARP3 and EP300, significantly inhibited apoptosis (Figures 5f and g and Supplementary Figure S8) and procaspase-3 activation (Figure 5h). Furthermore, miR-630 overexpression markedly decreased acetylated and phosphorylated p53 and total p53 in CIS-exposed A549 (Figure 5i). EP300 may act as a regulator of p53 via its acetylase.39,40 Therefore, decreased acetylation of p53 should be attributed to miR-630-targeted EP300. Our data suggest that miR-630 may directly or indirectly downregulate both apoptotic activator(s) and inhibitor(s) that have a bimodal role in the regulation of apoptosis. On the one hand, miR-630 promoted apoptosis by downregulating CDC7; on the other hand, it reduced apoptosis by downregulating apoptotic activators DDIT4, PARP3, EP300 and p53, thereby maintaining apoptotic balance in DDR (Figure 7). This can explain why we could not detect apoptosis in miR-630-transfected cells (Figure 5a). Although miR-630 transfection or induction may partly offset p53 acetylation and phosphorylation, the levels of chemomodified p53 in CIS-exposed cells were still higher than control (Figure 5i). We found recently that p53 was accumulated in HCT116p53+/+ and A549 upon CIS-induced DNA damage.54 Our recent observation54 and the data fromFigure 5i suggest that apoptotic mechanism still works in the presence of CIS, although miR-630 downregulates p53 by targeting EP300. Notably, miR-630 overexpression is an extreme case that differs from miR-630 induction by CIS. Therefore, the bimodal role of miR-630 does not discriminate against the contributions of CDC7 inactivation and other apoptotic regulators to CIS-induced apoptosis.

Bottom Line: We found that miR-630 was highly expressed in A549 and NIH3T3 cells where CDC7 was downregulated, but lower in H1299, MCF7, MDA-MB-231, HeLa and 2BS cells where CDC7 was upregulated.Our data also support the notion that a certain mRNA can be targeted by several miRNAs, and in particular an miRNA may target a set of mRNAs.These data afford a comprehensive view of microRNA-dependent control of gene expression in the regulation of apoptosis under genotoxic stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Xue Yuan Road 38, Beijing 100191, People's Republic of China.

ABSTRACT
MicroRNAome analyses have shown microRNA-630 (miR-630) to be involved in the regulation of apoptosis. However, its apoptotic role is still debated and its participation in DNA replication is unknown. Here, we demonstrate that miR-630 inhibits cell proliferation by targeting cell-cycle kinase 7 (CDC7) kinase, but maintains the apoptotic balance by targeting multiple activators of apoptosis under genotoxic stress. We identified a novel regulatory mechanism of CDC7 gene expression, in which miR-630 downregulated CDC7 expression by recognizing and binding to four binding sites in CDC7 3'-UTR. We found that miR-630 was highly expressed in A549 and NIH3T3 cells where CDC7 was downregulated, but lower in H1299, MCF7, MDA-MB-231, HeLa and 2BS cells where CDC7 was upregulated. Furthermore, the induction of miR-630 occurred commonly in a variety of human cancer and immortalized cells in response to genotoxic agents. Importantly, downregulation of CDC7 by miR-630 was associated with cisplatin (CIS)-induced inhibitory proliferation in A549 cells. Mechanistically, miR-630 exerted its inhibitory proliferation by blocking CDC7-mediated initiation of DNA synthesis and by inducing G1 arrest, but maintains apoptotic balance under CIS exposure. On the one hand, miR-630 promoted apoptosis by downregulation of CDC7; on the other hand, it reduced apoptosis by downregulating several apoptotic modulators such as PARP3, DDIT4, EP300 and EP300 downstream effector p53, thereby maintaining the apoptotic balance. Our data indicate that miR-630 has a bimodal role in the regulation of apoptosis in response to DNA damage. Our data also support the notion that a certain mRNA can be targeted by several miRNAs, and in particular an miRNA may target a set of mRNAs. These data afford a comprehensive view of microRNA-dependent control of gene expression in the regulation of apoptosis under genotoxic stress.

Show MeSH
Related in: MedlinePlus