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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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MiR-630 downregulates CDC7 expression by targeting CDC7 3'-UTR. A549 cells were transfected with miR-630 mimic or an inhibitor (50 nM) for 48 h. (a) RT-qPCR for CDC7 mRNA downregulation by miR-630. CDC7 mRNA was quantified by the 2−ΔΔCt method, in which glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The scrambled small interfering RNA (siRNA) was used as a negative control, under which condition the level of CDC7 mRNA was normalized to ‘1'. Data are presented as mean±S.D. (n=3). (b) Western blotting for CDC7 protein downregulation by miR-630. α-Tubulin was used as a loading control. (c) RT-qPCR and (d) western blotting for effects of miR-630 inhibitor on CDC7 mRNA and protein expression. Anti-scrambled siRNA was used as a control. Data are presented as mean±S.D. (n=3). (e) Western blotting for CDC7 protein in H1299, MCF7 and MDA-MB-231 cells transfected with miR-630 mimic for 48 h. (f) The predicted miR-630-binding sequences or mutated versions of CDC7 3'-UTR fragments ‘A', ‘B', ‘D' and ‘E' in (g). WT, wild type; MT, mutant (mutated bases are underlined). (g) Interpretation of luciferase reporter plasmids containing full-length CDC7 3'-UTR, fragments ‘A' to ‘E' or mutants (upper panel). The full-length 3'-UTR, truncates ‘A' to ‘E' and mutants in (f) were inserted into the pMIR-Report plasmid to generate pMIR-Report-PmiR-3'-UTR and its variations. ‘1' to ‘4' indicates the miR-630 binding sites. (h) Relative luciferase activities of the reporter plasmids in A549 cells. Data are presented as mean±S.D. (n=3). *P<0.05, **P<0.01 and ***P<0.001
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fig1: MiR-630 downregulates CDC7 expression by targeting CDC7 3'-UTR. A549 cells were transfected with miR-630 mimic or an inhibitor (50 nM) for 48 h. (a) RT-qPCR for CDC7 mRNA downregulation by miR-630. CDC7 mRNA was quantified by the 2−ΔΔCt method, in which glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The scrambled small interfering RNA (siRNA) was used as a negative control, under which condition the level of CDC7 mRNA was normalized to ‘1'. Data are presented as mean±S.D. (n=3). (b) Western blotting for CDC7 protein downregulation by miR-630. α-Tubulin was used as a loading control. (c) RT-qPCR and (d) western blotting for effects of miR-630 inhibitor on CDC7 mRNA and protein expression. Anti-scrambled siRNA was used as a control. Data are presented as mean±S.D. (n=3). (e) Western blotting for CDC7 protein in H1299, MCF7 and MDA-MB-231 cells transfected with miR-630 mimic for 48 h. (f) The predicted miR-630-binding sequences or mutated versions of CDC7 3'-UTR fragments ‘A', ‘B', ‘D' and ‘E' in (g). WT, wild type; MT, mutant (mutated bases are underlined). (g) Interpretation of luciferase reporter plasmids containing full-length CDC7 3'-UTR, fragments ‘A' to ‘E' or mutants (upper panel). The full-length 3'-UTR, truncates ‘A' to ‘E' and mutants in (f) were inserted into the pMIR-Report plasmid to generate pMIR-Report-PmiR-3'-UTR and its variations. ‘1' to ‘4' indicates the miR-630 binding sites. (h) Relative luciferase activities of the reporter plasmids in A549 cells. Data are presented as mean±S.D. (n=3). *P<0.05, **P<0.01 and ***P<0.001

Mentions: Depletion of CDC7 induces apoptosis in cancer cells.15,16 MiR-630 may target BCL2, BCL2L2 and IGF-1R to induce apoptosis under genotoxic stresses.34,35 As an miRNA may have multiple targets,14,37 we speculated that miR-630-induced inhibitory proliferation and, perhaps, apoptosis might be linked to CDC7. To demonstrate this hypothesis, the potential targets of miR-630 were searched by TargetScan software (http://www.targetscan.org), and CDC7 was selected. To validate whether miR-630 could target CDC7, we performed real-time quantitative PCR (RT-qPCR) to check the transfection efficiency (Supplementary Figure S1) and CDC7 expression after transfection of miR-630 mimic and inhibitor into A549 (p53-wt) cells. RT-qPCR and western blotting revealed that compared with transfection of scrambled siRNA, transfection of miR-630 mimic caused marked decreases in CDC7 mRNA and protein (Figures 1a and b), whereas transfection of miR-630 inhibitor led to significant increases of CDC7 mRNA and protein (Figures 1c and d). CDC7 downregulation was also observed in miR-630 mimic-transfected H1299 (p53-), MCF7 (p53-wt) and MDA-MB-231 (p53-mutant) cells (Figure 1e). These data indicate that miR-630 may target and inhibit CDC7 in a cell-type-independent manner.


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 downregulates CDC7 expression by targeting CDC7 3'-UTR. A549 cells were transfected with miR-630 mimic or an inhibitor (50 nM) for 48 h. (a) RT-qPCR for CDC7 mRNA downregulation by miR-630. CDC7 mRNA was quantified by the 2−ΔΔCt method, in which glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The scrambled small interfering RNA (siRNA) was used as a negative control, under which condition the level of CDC7 mRNA was normalized to ‘1'. Data are presented as mean±S.D. (n=3). (b) Western blotting for CDC7 protein downregulation by miR-630. α-Tubulin was used as a loading control. (c) RT-qPCR and (d) western blotting for effects of miR-630 inhibitor on CDC7 mRNA and protein expression. Anti-scrambled siRNA was used as a control. Data are presented as mean±S.D. (n=3). (e) Western blotting for CDC7 protein in H1299, MCF7 and MDA-MB-231 cells transfected with miR-630 mimic for 48 h. (f) The predicted miR-630-binding sequences or mutated versions of CDC7 3'-UTR fragments ‘A', ‘B', ‘D' and ‘E' in (g). WT, wild type; MT, mutant (mutated bases are underlined). (g) Interpretation of luciferase reporter plasmids containing full-length CDC7 3'-UTR, fragments ‘A' to ‘E' or mutants (upper panel). The full-length 3'-UTR, truncates ‘A' to ‘E' and mutants in (f) were inserted into the pMIR-Report plasmid to generate pMIR-Report-PmiR-3'-UTR and its variations. ‘1' to ‘4' indicates the miR-630 binding sites. (h) Relative luciferase activities of the reporter plasmids in A549 cells. Data are presented as mean±S.D. (n=3). *P<0.05, **P<0.01 and ***P<0.001
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fig1: MiR-630 downregulates CDC7 expression by targeting CDC7 3'-UTR. A549 cells were transfected with miR-630 mimic or an inhibitor (50 nM) for 48 h. (a) RT-qPCR for CDC7 mRNA downregulation by miR-630. CDC7 mRNA was quantified by the 2−ΔΔCt method, in which glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The scrambled small interfering RNA (siRNA) was used as a negative control, under which condition the level of CDC7 mRNA was normalized to ‘1'. Data are presented as mean±S.D. (n=3). (b) Western blotting for CDC7 protein downregulation by miR-630. α-Tubulin was used as a loading control. (c) RT-qPCR and (d) western blotting for effects of miR-630 inhibitor on CDC7 mRNA and protein expression. Anti-scrambled siRNA was used as a control. Data are presented as mean±S.D. (n=3). (e) Western blotting for CDC7 protein in H1299, MCF7 and MDA-MB-231 cells transfected with miR-630 mimic for 48 h. (f) The predicted miR-630-binding sequences or mutated versions of CDC7 3'-UTR fragments ‘A', ‘B', ‘D' and ‘E' in (g). WT, wild type; MT, mutant (mutated bases are underlined). (g) Interpretation of luciferase reporter plasmids containing full-length CDC7 3'-UTR, fragments ‘A' to ‘E' or mutants (upper panel). The full-length 3'-UTR, truncates ‘A' to ‘E' and mutants in (f) were inserted into the pMIR-Report plasmid to generate pMIR-Report-PmiR-3'-UTR and its variations. ‘1' to ‘4' indicates the miR-630 binding sites. (h) Relative luciferase activities of the reporter plasmids in A549 cells. Data are presented as mean±S.D. (n=3). *P<0.05, **P<0.01 and ***P<0.001
Mentions: Depletion of CDC7 induces apoptosis in cancer cells.15,16 MiR-630 may target BCL2, BCL2L2 and IGF-1R to induce apoptosis under genotoxic stresses.34,35 As an miRNA may have multiple targets,14,37 we speculated that miR-630-induced inhibitory proliferation and, perhaps, apoptosis might be linked to CDC7. To demonstrate this hypothesis, the potential targets of miR-630 were searched by TargetScan software (http://www.targetscan.org), and CDC7 was selected. To validate whether miR-630 could target CDC7, we performed real-time quantitative PCR (RT-qPCR) to check the transfection efficiency (Supplementary Figure S1) and CDC7 expression after transfection of miR-630 mimic and inhibitor into A549 (p53-wt) cells. RT-qPCR and western blotting revealed that compared with transfection of scrambled siRNA, transfection of miR-630 mimic caused marked decreases in CDC7 mRNA and protein (Figures 1a and b), whereas transfection of miR-630 inhibitor led to significant increases of CDC7 mRNA and protein (Figures 1c and d). CDC7 downregulation was also observed in miR-630 mimic-transfected H1299 (p53-), MCF7 (p53-wt) and MDA-MB-231 (p53-mutant) cells (Figure 1e). These data indicate that miR-630 may target and inhibit CDC7 in a cell-type-independent manner.

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