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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 expression inversely correlates with CDC7 expression in a variety of cells. (a) Western blotting for CDC7 protein in A549, H1299, MCF7, MDA-MB-231, HeLa, NIH3T3 and 2BS cells. Cells were grown in culture for 48 h. Western blotting was performed with anti-CDC7 antibody. α-Tubulin was used as a loading control. (b) Stem-loop RT-PCR analysis of miR-630 in several cells. The expression levels of miR-630 were quantified by RT-qPCR and normalized to the levels of U6 snRNA. Data present mean±S.D. (n=3). (c) The correlation of CDC7 protein and miR-630 levels; r=−0.8735; P=0.0102. (d) Northern blotting for miR-630 expression in CIS-exposed A549. Cells were exposed to 100 μM CIS for 36 h and Northern blotting was performed. U1, 5SRNA and tRNA were used as loading controls. Unexposed cells were used as control (Con). (e) RT-qPCR for miR-630 expression in CIS-exposed A549. Data are presented as mean±S.D. (n=3); *P=0.0347. (f) RT-qPCR and (g) western blotting for CDC7 mRNA and protein in CIS-exposed A549. Data are presented as mean±S.D. (n=3); *P=0.0171. (h) The effect of anti-miR-630 on CDC7 expression. A549 cells were transfected with miR-630 inhibitor (50 nM) for 48 h, and exposed to CIS for 36 h, followed by western blotting with anti-CDC7 antibody. The blots were screened/quantified and normalized against β-actin level. The value obtained from scrambled small interfering RNA (siRNA)/CIS-unexposed cells was designated as ‘1' (lane 1, bottom). (i and j) The effects of p53 transfection on miR-630 and CDC7 expression. A549 (p53-wild type) and H1299 (p53-) cells were transfected with pcDNA3.1-p53 (pcDNA3.1 as control) for 48 h, followed by RT-qPCR for miR-630 (i) and western blotting for CDC7 and p53 (j). Data are presented as mean±S.D. (n=3) in (i), and α-tubulin was used as a loading control in (j). (k) P53 status in A549 and H1299. Cells were grown and exposed or unexposed to CIS for 36 h, followed by western blotting for p53 and p21 expression, and β-actin was used as a loading control
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fig2: MiR-630 expression inversely correlates with CDC7 expression in a variety of cells. (a) Western blotting for CDC7 protein in A549, H1299, MCF7, MDA-MB-231, HeLa, NIH3T3 and 2BS cells. Cells were grown in culture for 48 h. Western blotting was performed with anti-CDC7 antibody. α-Tubulin was used as a loading control. (b) Stem-loop RT-PCR analysis of miR-630 in several cells. The expression levels of miR-630 were quantified by RT-qPCR and normalized to the levels of U6 snRNA. Data present mean±S.D. (n=3). (c) The correlation of CDC7 protein and miR-630 levels; r=−0.8735; P=0.0102. (d) Northern blotting for miR-630 expression in CIS-exposed A549. Cells were exposed to 100 μM CIS for 36 h and Northern blotting was performed. U1, 5SRNA and tRNA were used as loading controls. Unexposed cells were used as control (Con). (e) RT-qPCR for miR-630 expression in CIS-exposed A549. Data are presented as mean±S.D. (n=3); *P=0.0347. (f) RT-qPCR and (g) western blotting for CDC7 mRNA and protein in CIS-exposed A549. Data are presented as mean±S.D. (n=3); *P=0.0171. (h) The effect of anti-miR-630 on CDC7 expression. A549 cells were transfected with miR-630 inhibitor (50 nM) for 48 h, and exposed to CIS for 36 h, followed by western blotting with anti-CDC7 antibody. The blots were screened/quantified and normalized against β-actin level. The value obtained from scrambled small interfering RNA (siRNA)/CIS-unexposed cells was designated as ‘1' (lane 1, bottom). (i and j) The effects of p53 transfection on miR-630 and CDC7 expression. A549 (p53-wild type) and H1299 (p53-) cells were transfected with pcDNA3.1-p53 (pcDNA3.1 as control) for 48 h, followed by RT-qPCR for miR-630 (i) and western blotting for CDC7 and p53 (j). Data are presented as mean±S.D. (n=3) in (i), and α-tubulin was used as a loading control in (j). (k) P53 status in A549 and H1299. Cells were grown and exposed or unexposed to CIS for 36 h, followed by western blotting for p53 and p21 expression, and β-actin was used as a loading control

Mentions: To study the correlation of CDC7 and miR-630 in cells, CDC7 and miR-630 expression in several cell lines was examined. CDC7 was expressed relatively lower or undetectable in A549 and NIH3T3, but high in H1299, MCF7, MDA-MB-231, HeLa and 2BS cells (Figure 2a). Contrarily, miR-630 was expressed very high in A549 and NIH3T3, but lower in H1299, MCF7, MDA-MB-231, HeLa and 2BS (Figure 2b). The scatter plot for CDC7 versus miR-630 revealed an inverse correlation of CDC7 and miR-630 in these cells (r=−0.8735, P=0.0102) (Figure 2c). These data indicate again that miR-630 acts as a modulator of CDC7. These data also suggest that miR-630 as well as CDC7 is differentially expressed in a variety of cancer and immortalized cells.


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 expression inversely correlates with CDC7 expression in a variety of cells. (a) Western blotting for CDC7 protein in A549, H1299, MCF7, MDA-MB-231, HeLa, NIH3T3 and 2BS cells. Cells were grown in culture for 48 h. Western blotting was performed with anti-CDC7 antibody. α-Tubulin was used as a loading control. (b) Stem-loop RT-PCR analysis of miR-630 in several cells. The expression levels of miR-630 were quantified by RT-qPCR and normalized to the levels of U6 snRNA. Data present mean±S.D. (n=3). (c) The correlation of CDC7 protein and miR-630 levels; r=−0.8735; P=0.0102. (d) Northern blotting for miR-630 expression in CIS-exposed A549. Cells were exposed to 100 μM CIS for 36 h and Northern blotting was performed. U1, 5SRNA and tRNA were used as loading controls. Unexposed cells were used as control (Con). (e) RT-qPCR for miR-630 expression in CIS-exposed A549. Data are presented as mean±S.D. (n=3); *P=0.0347. (f) RT-qPCR and (g) western blotting for CDC7 mRNA and protein in CIS-exposed A549. Data are presented as mean±S.D. (n=3); *P=0.0171. (h) The effect of anti-miR-630 on CDC7 expression. A549 cells were transfected with miR-630 inhibitor (50 nM) for 48 h, and exposed to CIS for 36 h, followed by western blotting with anti-CDC7 antibody. The blots were screened/quantified and normalized against β-actin level. The value obtained from scrambled small interfering RNA (siRNA)/CIS-unexposed cells was designated as ‘1' (lane 1, bottom). (i and j) The effects of p53 transfection on miR-630 and CDC7 expression. A549 (p53-wild type) and H1299 (p53-) cells were transfected with pcDNA3.1-p53 (pcDNA3.1 as control) for 48 h, followed by RT-qPCR for miR-630 (i) and western blotting for CDC7 and p53 (j). Data are presented as mean±S.D. (n=3) in (i), and α-tubulin was used as a loading control in (j). (k) P53 status in A549 and H1299. Cells were grown and exposed or unexposed to CIS for 36 h, followed by western blotting for p53 and p21 expression, and β-actin was used as a loading control
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Related In: Results  -  Collection

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fig2: MiR-630 expression inversely correlates with CDC7 expression in a variety of cells. (a) Western blotting for CDC7 protein in A549, H1299, MCF7, MDA-MB-231, HeLa, NIH3T3 and 2BS cells. Cells were grown in culture for 48 h. Western blotting was performed with anti-CDC7 antibody. α-Tubulin was used as a loading control. (b) Stem-loop RT-PCR analysis of miR-630 in several cells. The expression levels of miR-630 were quantified by RT-qPCR and normalized to the levels of U6 snRNA. Data present mean±S.D. (n=3). (c) The correlation of CDC7 protein and miR-630 levels; r=−0.8735; P=0.0102. (d) Northern blotting for miR-630 expression in CIS-exposed A549. Cells were exposed to 100 μM CIS for 36 h and Northern blotting was performed. U1, 5SRNA and tRNA were used as loading controls. Unexposed cells were used as control (Con). (e) RT-qPCR for miR-630 expression in CIS-exposed A549. Data are presented as mean±S.D. (n=3); *P=0.0347. (f) RT-qPCR and (g) western blotting for CDC7 mRNA and protein in CIS-exposed A549. Data are presented as mean±S.D. (n=3); *P=0.0171. (h) The effect of anti-miR-630 on CDC7 expression. A549 cells were transfected with miR-630 inhibitor (50 nM) for 48 h, and exposed to CIS for 36 h, followed by western blotting with anti-CDC7 antibody. The blots were screened/quantified and normalized against β-actin level. The value obtained from scrambled small interfering RNA (siRNA)/CIS-unexposed cells was designated as ‘1' (lane 1, bottom). (i and j) The effects of p53 transfection on miR-630 and CDC7 expression. A549 (p53-wild type) and H1299 (p53-) cells were transfected with pcDNA3.1-p53 (pcDNA3.1 as control) for 48 h, followed by RT-qPCR for miR-630 (i) and western blotting for CDC7 and p53 (j). Data are presented as mean±S.D. (n=3) in (i), and α-tubulin was used as a loading control in (j). (k) P53 status in A549 and H1299. Cells were grown and exposed or unexposed to CIS for 36 h, followed by western blotting for p53 and p21 expression, and β-actin was used as a loading control
Mentions: To study the correlation of CDC7 and miR-630 in cells, CDC7 and miR-630 expression in several cell lines was examined. CDC7 was expressed relatively lower or undetectable in A549 and NIH3T3, but high in H1299, MCF7, MDA-MB-231, HeLa and 2BS cells (Figure 2a). Contrarily, miR-630 was expressed very high in A549 and NIH3T3, but lower in H1299, MCF7, MDA-MB-231, HeLa and 2BS (Figure 2b). The scatter plot for CDC7 versus miR-630 revealed an inverse correlation of CDC7 and miR-630 in these cells (r=−0.8735, P=0.0102) (Figure 2c). These data indicate again that miR-630 acts as a modulator of CDC7. These data also suggest that miR-630 as well as CDC7 is differentially expressed in a variety of cancer and immortalized cells.

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