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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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Induction of miR-630 is associated with CIS-induced G1 arrest. A549 cells were synchronized at G0/G1 phase by serum starvation for 48 h, re-feeding with 20% FBS and treated with CIS for 0, 3, 6, 12, 24 and 36 h, respectively. (a) A representative of flow cytometry showing CIS induced G1 arrest. (b) Histograms showing the populations of the cell-cycle in (a) experiments. Cells (2 × l05) were fixed and stained with PI, and analyzed by FACScan. Data present mean from two independent experiments. (c) The expression of miR-630 in (a) experiments. MiR-630 was determined by RT-qPCR and U6 was used as internal control. Data present mean±S.D. (n=3). (d) Western blotting for ATM/p-ATM, ATR/p-ATR, CDC7, p53, p21, cyclin D1, p38/p-p38 in (a) experiments. α-Tubulin as loading control. (e) G1 arrest in miR-630 mimic-transfected A549 cells. Cells were transfected with miR-630 mimic (scrambled small interfering RNA (siRNA) as control) for 48 h, followed by analysis of the cell-cycle (for CDC7 and p-p38 expression see Figure 5b, right panel)
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fig6: Induction of miR-630 is associated with CIS-induced G1 arrest. A549 cells were synchronized at G0/G1 phase by serum starvation for 48 h, re-feeding with 20% FBS and treated with CIS for 0, 3, 6, 12, 24 and 36 h, respectively. (a) A representative of flow cytometry showing CIS induced G1 arrest. (b) Histograms showing the populations of the cell-cycle in (a) experiments. Cells (2 × l05) were fixed and stained with PI, and analyzed by FACScan. Data present mean from two independent experiments. (c) The expression of miR-630 in (a) experiments. MiR-630 was determined by RT-qPCR and U6 was used as internal control. Data present mean±S.D. (n=3). (d) Western blotting for ATM/p-ATM, ATR/p-ATR, CDC7, p53, p21, cyclin D1, p38/p-p38 in (a) experiments. α-Tubulin as loading control. (e) G1 arrest in miR-630 mimic-transfected A549 cells. Cells were transfected with miR-630 mimic (scrambled small interfering RNA (siRNA) as control) for 48 h, followed by analysis of the cell-cycle (for CDC7 and p-p38 expression see Figure 5b, right panel)

Mentions: To investigate the effects of CIS-induced miR-630 on cell cycle, A549 cells were synchronized at G0/G1-phase by serum starvation and released into the cell cycle by adding 20% fetal calf serum and exposed to CIS for given hours, followed by cell-cycle analysis. G1 arrest was observed immediately after CIS exposure and sustained until at least 36 h (Figures 6a and b). The majority of G1 population with a sub-G1 peak appeared 24 h after exposure (Figure 6a). Meanwhile, miR-630 was upregulated within 3–36 h after exposure (Figure 6c). Contrarily, CDC7 was markedly downregulated after 6 h exposure (Figure 6d). Supporting G1 arrest, CIS exposure increased p-ATM, p53 and p21 expression, whereas cyclin D1 was downregulated after 6 h exposure. Phospho-ATR was consistently detectable, although its levels were not increased. Moreover, phospho-p38 was increased (Figure 6d). These data indicate that CIS exposure may induce G1 arrest and apoptosis in A549. It seems likely that CIS-induced G1 arrest is attributed to the activation of ATM/ATR-p53-p21 signaling pathway, and CIS-induced apoptosis is linked to the activation of p53 and p38 kinase.


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)

Induction of miR-630 is associated with CIS-induced G1 arrest. A549 cells were synchronized at G0/G1 phase by serum starvation for 48 h, re-feeding with 20% FBS and treated with CIS for 0, 3, 6, 12, 24 and 36 h, respectively. (a) A representative of flow cytometry showing CIS induced G1 arrest. (b) Histograms showing the populations of the cell-cycle in (a) experiments. Cells (2 × l05) were fixed and stained with PI, and analyzed by FACScan. Data present mean from two independent experiments. (c) The expression of miR-630 in (a) experiments. MiR-630 was determined by RT-qPCR and U6 was used as internal control. Data present mean±S.D. (n=3). (d) Western blotting for ATM/p-ATM, ATR/p-ATR, CDC7, p53, p21, cyclin D1, p38/p-p38 in (a) experiments. α-Tubulin as loading control. (e) G1 arrest in miR-630 mimic-transfected A549 cells. Cells were transfected with miR-630 mimic (scrambled small interfering RNA (siRNA) as control) for 48 h, followed by analysis of the cell-cycle (for CDC7 and p-p38 expression see Figure 5b, right panel)
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fig6: Induction of miR-630 is associated with CIS-induced G1 arrest. A549 cells were synchronized at G0/G1 phase by serum starvation for 48 h, re-feeding with 20% FBS and treated with CIS for 0, 3, 6, 12, 24 and 36 h, respectively. (a) A representative of flow cytometry showing CIS induced G1 arrest. (b) Histograms showing the populations of the cell-cycle in (a) experiments. Cells (2 × l05) were fixed and stained with PI, and analyzed by FACScan. Data present mean from two independent experiments. (c) The expression of miR-630 in (a) experiments. MiR-630 was determined by RT-qPCR and U6 was used as internal control. Data present mean±S.D. (n=3). (d) Western blotting for ATM/p-ATM, ATR/p-ATR, CDC7, p53, p21, cyclin D1, p38/p-p38 in (a) experiments. α-Tubulin as loading control. (e) G1 arrest in miR-630 mimic-transfected A549 cells. Cells were transfected with miR-630 mimic (scrambled small interfering RNA (siRNA) as control) for 48 h, followed by analysis of the cell-cycle (for CDC7 and p-p38 expression see Figure 5b, right panel)
Mentions: To investigate the effects of CIS-induced miR-630 on cell cycle, A549 cells were synchronized at G0/G1-phase by serum starvation and released into the cell cycle by adding 20% fetal calf serum and exposed to CIS for given hours, followed by cell-cycle analysis. G1 arrest was observed immediately after CIS exposure and sustained until at least 36 h (Figures 6a and b). The majority of G1 population with a sub-G1 peak appeared 24 h after exposure (Figure 6a). Meanwhile, miR-630 was upregulated within 3–36 h after exposure (Figure 6c). Contrarily, CDC7 was markedly downregulated after 6 h exposure (Figure 6d). Supporting G1 arrest, CIS exposure increased p-ATM, p53 and p21 expression, whereas cyclin D1 was downregulated after 6 h exposure. Phospho-ATR was consistently detectable, although its levels were not increased. Moreover, phospho-p38 was increased (Figure 6d). These data indicate that CIS exposure may induce G1 arrest and apoptosis in A549. It seems likely that CIS-induced G1 arrest is attributed to the activation of ATM/ATR-p53-p21 signaling pathway, and CIS-induced apoptosis is linked to the activation of p53 and p38 kinase.

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