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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 maintains the apoptotic balance by targeting multiple apoptotic regulators. (a) Annexin V/PI assay for apoptosis in A549 cells. Cells were transfected with scrambled small interfering RNA (siRNA), miR-630 mimic or inhibitor for 48 h. After double staining with Annexin V/PI, flow cytometry were performed. Data are presented mean±S.D. (n=5). For flow cytometry dot plots see Supplementary Figure S6. (b) Activation of p38 kinase by miR-630 transfection or silencing CDC7. A549 cells were transfected with scrambled siRNA, miR-630 mimic or CDC7 siRNA (siCDC7-1) for 48 h, followed by western blotting for CDC7 and p-p38 expression with specific antibodies. α-Tubulin was used as a loading control. (c) Relative luciferase activities of the reporter plasmids. Luciferase reporter constructs were co-transfected with scrambled siRNA or miR-630 mimic into A549. Luciferase reporter activities were assayed 48 h after transfection and normalized to scrambled siRNA. Data are presented as mean±S.D. (n=3). (d) The effects of miR-630 on DDIT4, PARP3 and EP300 mRNA expression. A549 cells were transfected with scrambled siRNA, miR-630 mimic (upper panel) and inhibitor (lower panel) for 48 h, followed by RT-qPCR for DDIT4, PARP3 and EP300 mRNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was an internal control. Data are presented as mean±S.D. (n=3). (e) The effects of miR-630 on DDIT4, PARP3 and EP300 protein expression. Transfection of A549 was described in (d), and PARP3, EP300 and DDIT4 proteins were analyzed by western blotting. α-Tubulin was used as a loading control. (f) and (g) Reduction of apoptosis by silencing DDIT4, PARP3 and EP300. DDIT4, PARP3 and EP300 were individually or jointly silenced by transfection of specific siRNA oligonucleotides into A549 cells, and apoptosis was examined 48 h after transfection. Data are presented as mean±S.D. (n=3). (f) A representative of flow cytometry for apoptosis induced by combined silencing of DDIT4, PARP3 and EP300. For all dot plots in (g) see Supplementary Figure S8. (h) Western blotting for activated caspase-3 (p17), PARP (p89) and silenced DDIT4, PARP3 and EP300 in (g) experiments. (i) Downregulation of CIS-induced p53 and its modifications by miR-630. A549 cells were transfected with scrambled siRNA or miR-630 mimic for 48 h, and exposed to CIS for additional 36 h. The acylation of p53 at Lys382 and phosphorylation of p53 at Ser15, Ser20 and Ser46 were examined by western blotting. β-Actin was used as a loading control. *P<0.05 and **P<0.01
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fig5: MiR-630 maintains the apoptotic balance by targeting multiple apoptotic regulators. (a) Annexin V/PI assay for apoptosis in A549 cells. Cells were transfected with scrambled small interfering RNA (siRNA), miR-630 mimic or inhibitor for 48 h. After double staining with Annexin V/PI, flow cytometry were performed. Data are presented mean±S.D. (n=5). For flow cytometry dot plots see Supplementary Figure S6. (b) Activation of p38 kinase by miR-630 transfection or silencing CDC7. A549 cells were transfected with scrambled siRNA, miR-630 mimic or CDC7 siRNA (siCDC7-1) for 48 h, followed by western blotting for CDC7 and p-p38 expression with specific antibodies. α-Tubulin was used as a loading control. (c) Relative luciferase activities of the reporter plasmids. Luciferase reporter constructs were co-transfected with scrambled siRNA or miR-630 mimic into A549. Luciferase reporter activities were assayed 48 h after transfection and normalized to scrambled siRNA. Data are presented as mean±S.D. (n=3). (d) The effects of miR-630 on DDIT4, PARP3 and EP300 mRNA expression. A549 cells were transfected with scrambled siRNA, miR-630 mimic (upper panel) and inhibitor (lower panel) for 48 h, followed by RT-qPCR for DDIT4, PARP3 and EP300 mRNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was an internal control. Data are presented as mean±S.D. (n=3). (e) The effects of miR-630 on DDIT4, PARP3 and EP300 protein expression. Transfection of A549 was described in (d), and PARP3, EP300 and DDIT4 proteins were analyzed by western blotting. α-Tubulin was used as a loading control. (f) and (g) Reduction of apoptosis by silencing DDIT4, PARP3 and EP300. DDIT4, PARP3 and EP300 were individually or jointly silenced by transfection of specific siRNA oligonucleotides into A549 cells, and apoptosis was examined 48 h after transfection. Data are presented as mean±S.D. (n=3). (f) A representative of flow cytometry for apoptosis induced by combined silencing of DDIT4, PARP3 and EP300. For all dot plots in (g) see Supplementary Figure S8. (h) Western blotting for activated caspase-3 (p17), PARP (p89) and silenced DDIT4, PARP3 and EP300 in (g) experiments. (i) Downregulation of CIS-induced p53 and its modifications by miR-630. A549 cells were transfected with scrambled siRNA or miR-630 mimic for 48 h, and exposed to CIS for additional 36 h. The acylation of p53 at Lys382 and phosphorylation of p53 at Ser15, Ser20 and Ser46 were examined by western blotting. β-Actin was used as a loading control. *P<0.05 and **P<0.01

Mentions: To determine the effects of miR-630-downregulated CDC7 on CIS-induced apoptosis, miR-630 mimic or inhibitor was transfected into A549, followed by the detection of apoptosis. Unexpectedly, in Annexin V/PI assays we could not detect any apoptosis in miR-630 mimic-transfected A549 compared with scrambled siRNA and inhibitor transfection (Figure 5a and Supplementary Figure S6), although miR-630 mimic as well as CDC7 siRNA markedly repressed CDC7 expression (Figure 5b). Consistently, procaspase-3 activation was undetectable (data not shown) in miR-630 mimic-transfected A549, indicating that miR-630 overexpression failed to induce apoptosis. As apoptosis induced by CDC7 depletion in cancer cells is associated with ATR-activated p38 kinase,16 we examined phosphorylation/activation of p38 kinase in CDC7-silenced A549 cells, where phosphor-p38 was upregulated (Figure 5b, left panel). Surprisingly, the activated kinase was also detectable in miR-630 mimic-transfected A549 (right panel). These results suggest that the p38 kinase-related apoptotic pathway is still operative in miR-630-expressed or CDC7-silenced A549. Thus, the functional significance of miR-630 induction in CIS-induced apoptosis remains to be established.


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 maintains the apoptotic balance by targeting multiple apoptotic regulators. (a) Annexin V/PI assay for apoptosis in A549 cells. Cells were transfected with scrambled small interfering RNA (siRNA), miR-630 mimic or inhibitor for 48 h. After double staining with Annexin V/PI, flow cytometry were performed. Data are presented mean±S.D. (n=5). For flow cytometry dot plots see Supplementary Figure S6. (b) Activation of p38 kinase by miR-630 transfection or silencing CDC7. A549 cells were transfected with scrambled siRNA, miR-630 mimic or CDC7 siRNA (siCDC7-1) for 48 h, followed by western blotting for CDC7 and p-p38 expression with specific antibodies. α-Tubulin was used as a loading control. (c) Relative luciferase activities of the reporter plasmids. Luciferase reporter constructs were co-transfected with scrambled siRNA or miR-630 mimic into A549. Luciferase reporter activities were assayed 48 h after transfection and normalized to scrambled siRNA. Data are presented as mean±S.D. (n=3). (d) The effects of miR-630 on DDIT4, PARP3 and EP300 mRNA expression. A549 cells were transfected with scrambled siRNA, miR-630 mimic (upper panel) and inhibitor (lower panel) for 48 h, followed by RT-qPCR for DDIT4, PARP3 and EP300 mRNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was an internal control. Data are presented as mean±S.D. (n=3). (e) The effects of miR-630 on DDIT4, PARP3 and EP300 protein expression. Transfection of A549 was described in (d), and PARP3, EP300 and DDIT4 proteins were analyzed by western blotting. α-Tubulin was used as a loading control. (f) and (g) Reduction of apoptosis by silencing DDIT4, PARP3 and EP300. DDIT4, PARP3 and EP300 were individually or jointly silenced by transfection of specific siRNA oligonucleotides into A549 cells, and apoptosis was examined 48 h after transfection. Data are presented as mean±S.D. (n=3). (f) A representative of flow cytometry for apoptosis induced by combined silencing of DDIT4, PARP3 and EP300. For all dot plots in (g) see Supplementary Figure S8. (h) Western blotting for activated caspase-3 (p17), PARP (p89) and silenced DDIT4, PARP3 and EP300 in (g) experiments. (i) Downregulation of CIS-induced p53 and its modifications by miR-630. A549 cells were transfected with scrambled siRNA or miR-630 mimic for 48 h, and exposed to CIS for additional 36 h. The acylation of p53 at Lys382 and phosphorylation of p53 at Ser15, Ser20 and Ser46 were examined by western blotting. β-Actin was used as a loading control. *P<0.05 and **P<0.01
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fig5: MiR-630 maintains the apoptotic balance by targeting multiple apoptotic regulators. (a) Annexin V/PI assay for apoptosis in A549 cells. Cells were transfected with scrambled small interfering RNA (siRNA), miR-630 mimic or inhibitor for 48 h. After double staining with Annexin V/PI, flow cytometry were performed. Data are presented mean±S.D. (n=5). For flow cytometry dot plots see Supplementary Figure S6. (b) Activation of p38 kinase by miR-630 transfection or silencing CDC7. A549 cells were transfected with scrambled siRNA, miR-630 mimic or CDC7 siRNA (siCDC7-1) for 48 h, followed by western blotting for CDC7 and p-p38 expression with specific antibodies. α-Tubulin was used as a loading control. (c) Relative luciferase activities of the reporter plasmids. Luciferase reporter constructs were co-transfected with scrambled siRNA or miR-630 mimic into A549. Luciferase reporter activities were assayed 48 h after transfection and normalized to scrambled siRNA. Data are presented as mean±S.D. (n=3). (d) The effects of miR-630 on DDIT4, PARP3 and EP300 mRNA expression. A549 cells were transfected with scrambled siRNA, miR-630 mimic (upper panel) and inhibitor (lower panel) for 48 h, followed by RT-qPCR for DDIT4, PARP3 and EP300 mRNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was an internal control. Data are presented as mean±S.D. (n=3). (e) The effects of miR-630 on DDIT4, PARP3 and EP300 protein expression. Transfection of A549 was described in (d), and PARP3, EP300 and DDIT4 proteins were analyzed by western blotting. α-Tubulin was used as a loading control. (f) and (g) Reduction of apoptosis by silencing DDIT4, PARP3 and EP300. DDIT4, PARP3 and EP300 were individually or jointly silenced by transfection of specific siRNA oligonucleotides into A549 cells, and apoptosis was examined 48 h after transfection. Data are presented as mean±S.D. (n=3). (f) A representative of flow cytometry for apoptosis induced by combined silencing of DDIT4, PARP3 and EP300. For all dot plots in (g) see Supplementary Figure S8. (h) Western blotting for activated caspase-3 (p17), PARP (p89) and silenced DDIT4, PARP3 and EP300 in (g) experiments. (i) Downregulation of CIS-induced p53 and its modifications by miR-630. A549 cells were transfected with scrambled siRNA or miR-630 mimic for 48 h, and exposed to CIS for additional 36 h. The acylation of p53 at Lys382 and phosphorylation of p53 at Ser15, Ser20 and Ser46 were examined by western blotting. β-Actin was used as a loading control. *P<0.05 and **P<0.01
Mentions: To determine the effects of miR-630-downregulated CDC7 on CIS-induced apoptosis, miR-630 mimic or inhibitor was transfected into A549, followed by the detection of apoptosis. Unexpectedly, in Annexin V/PI assays we could not detect any apoptosis in miR-630 mimic-transfected A549 compared with scrambled siRNA and inhibitor transfection (Figure 5a and Supplementary Figure S6), although miR-630 mimic as well as CDC7 siRNA markedly repressed CDC7 expression (Figure 5b). Consistently, procaspase-3 activation was undetectable (data not shown) in miR-630 mimic-transfected A549, indicating that miR-630 overexpression failed to induce apoptosis. As apoptosis induced by CDC7 depletion in cancer cells is associated with ATR-activated p38 kinase,16 we examined phosphorylation/activation of p38 kinase in CDC7-silenced A549 cells, where phosphor-p38 was upregulated (Figure 5b, left panel). Surprisingly, the activated kinase was also detectable in miR-630 mimic-transfected A549 (right panel). These results suggest that the p38 kinase-related apoptotic pathway is still operative in miR-630-expressed or CDC7-silenced A549. Thus, the functional significance of miR-630 induction in CIS-induced apoptosis remains to be established.

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