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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.Importantly, downregulation of CDC7 by miR-630 was associated with cisplatin (CIS)-induced inhibitory proliferation in A549 cells.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.

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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MiR-630 inhibits cell proliferation by inhibiting CDC7-mediated DNA synthesis. A549 cells were transfected with scrambled small interfering RNA (siRNA) (control), miR-630 mimic and miR-630 inhibitor for 48 and 72 h. (a) Western blotting for CDC7, MCM2 and phospho-MCM2. β-Actin was used as a loading control. (b) Flow cytometric analysis of BrdU-positive cells. After 48 and 72 h transfection, cells were labeled with 50 μM BrdU for 1 h before collection. Samples were stained with anti-BrdU FITC antibody and PI, and analyzed by flow cytometry. BrdU-positive cells were included in the gate region. (c) Relative amount of BrdU-positive cells in the gate region compared with total cells in (b) experiments. Data are presented as mean±S.D. (n=3); 48 h: *P=0.0396, ##P=0.0054; 72 h: **P=0.0037, #P=0.0171. (d) MTS assay showed the survival of A549 cells transfected with miR-630 mimic or inhibitor for 48 h. Data are presented as mean±S.D. (n=3); **P=0.0026 and #P=0.0223
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fig4: MiR-630 inhibits cell proliferation by inhibiting CDC7-mediated DNA synthesis. A549 cells were transfected with scrambled small interfering RNA (siRNA) (control), miR-630 mimic and miR-630 inhibitor for 48 and 72 h. (a) Western blotting for CDC7, MCM2 and phospho-MCM2. β-Actin was used as a loading control. (b) Flow cytometric analysis of BrdU-positive cells. After 48 and 72 h transfection, cells were labeled with 50 μM BrdU for 1 h before collection. Samples were stained with anti-BrdU FITC antibody and PI, and analyzed by flow cytometry. BrdU-positive cells were included in the gate region. (c) Relative amount of BrdU-positive cells in the gate region compared with total cells in (b) experiments. Data are presented as mean±S.D. (n=3); 48 h: *P=0.0396, ##P=0.0054; 72 h: **P=0.0037, #P=0.0171. (d) MTS assay showed the survival of A549 cells transfected with miR-630 mimic or inhibitor for 48 h. Data are presented as mean±S.D. (n=3); **P=0.0026 and #P=0.0223

Mentions: MCM2 phosphorylation by CDC7 promotes binding of CDC45 to the pre-RC during replisome assembly.3,7 To study the effects of CDC7 downregulation by miR-630 on DNA replication initiation and progression, miR-630 mimic or inhibitor was transfected into A549. After 48 and 72 h transfection, CDC7 and phosphorylated MCM2 were downregulated by miR-630 mimic compared with miR-630 inhibitor and scrambled siRNA (Figure 4a). To analyze DNA synthesis, transfected cells were labeled with bromo-deoxyuridine (BrdU) and double-stained with anti-BrdU antibody and propidium iodide (PI), followed by flow cytometry assays (Figure 4b). Compared with scrambled siRNA-transfected cells, the number of BrdU-positive cells increased 3.9% and 4.8% at 48 and 72 h in miR-630 inhibitor-transfected cells (P=0.0054; P=0.0171), but reduced 3.3% and 7.8% in miR-630 mimic-transfected cells (P=0.0396; P=0.0037), respectively (Figure 4c). Consistently, miR-630 mimic reduced cell survival by ~23% after 48 h transfection, whereas miR-630 inhibitor increased survival by 22.2% (Figure 4d). These results suggest that inhibitory proliferation of A549 cells by miR-630 is attributed to the inhibition of CDC7-mediated DNA synthesis.


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)

MiR-630 inhibits cell proliferation by inhibiting CDC7-mediated DNA synthesis. A549 cells were transfected with scrambled small interfering RNA (siRNA) (control), miR-630 mimic and miR-630 inhibitor for 48 and 72 h. (a) Western blotting for CDC7, MCM2 and phospho-MCM2. β-Actin was used as a loading control. (b) Flow cytometric analysis of BrdU-positive cells. After 48 and 72 h transfection, cells were labeled with 50 μM BrdU for 1 h before collection. Samples were stained with anti-BrdU FITC antibody and PI, and analyzed by flow cytometry. BrdU-positive cells were included in the gate region. (c) Relative amount of BrdU-positive cells in the gate region compared with total cells in (b) experiments. Data are presented as mean±S.D. (n=3); 48 h: *P=0.0396, ##P=0.0054; 72 h: **P=0.0037, #P=0.0171. (d) MTS assay showed the survival of A549 cells transfected with miR-630 mimic or inhibitor for 48 h. Data are presented as mean±S.D. (n=3); **P=0.0026 and #P=0.0223
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fig4: MiR-630 inhibits cell proliferation by inhibiting CDC7-mediated DNA synthesis. A549 cells were transfected with scrambled small interfering RNA (siRNA) (control), miR-630 mimic and miR-630 inhibitor for 48 and 72 h. (a) Western blotting for CDC7, MCM2 and phospho-MCM2. β-Actin was used as a loading control. (b) Flow cytometric analysis of BrdU-positive cells. After 48 and 72 h transfection, cells were labeled with 50 μM BrdU for 1 h before collection. Samples were stained with anti-BrdU FITC antibody and PI, and analyzed by flow cytometry. BrdU-positive cells were included in the gate region. (c) Relative amount of BrdU-positive cells in the gate region compared with total cells in (b) experiments. Data are presented as mean±S.D. (n=3); 48 h: *P=0.0396, ##P=0.0054; 72 h: **P=0.0037, #P=0.0171. (d) MTS assay showed the survival of A549 cells transfected with miR-630 mimic or inhibitor for 48 h. Data are presented as mean±S.D. (n=3); **P=0.0026 and #P=0.0223
Mentions: MCM2 phosphorylation by CDC7 promotes binding of CDC45 to the pre-RC during replisome assembly.3,7 To study the effects of CDC7 downregulation by miR-630 on DNA replication initiation and progression, miR-630 mimic or inhibitor was transfected into A549. After 48 and 72 h transfection, CDC7 and phosphorylated MCM2 were downregulated by miR-630 mimic compared with miR-630 inhibitor and scrambled siRNA (Figure 4a). To analyze DNA synthesis, transfected cells were labeled with bromo-deoxyuridine (BrdU) and double-stained with anti-BrdU antibody and propidium iodide (PI), followed by flow cytometry assays (Figure 4b). Compared with scrambled siRNA-transfected cells, the number of BrdU-positive cells increased 3.9% and 4.8% at 48 and 72 h in miR-630 inhibitor-transfected cells (P=0.0054; P=0.0171), but reduced 3.3% and 7.8% in miR-630 mimic-transfected cells (P=0.0396; P=0.0037), respectively (Figure 4c). Consistently, miR-630 mimic reduced cell survival by ~23% after 48 h transfection, whereas miR-630 inhibitor increased survival by 22.2% (Figure 4d). These results suggest that inhibitory proliferation of A549 cells by miR-630 is attributed to the inhibition of CDC7-mediated DNA synthesis.

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.Importantly, downregulation of CDC7 by miR-630 was associated with cisplatin (CIS)-induced inhibitory proliferation in A549 cells.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.

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