Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jun;67(12):2069-76.
doi: 10.1007/s00018-010-0340-8. Epub 2010 Mar 19.

Aurora kinase A induces miR-17-92 cluster through regulation of E2F1 transcription factor

Shun He  1 , Shangbin Yang, Guohua Deng, Mei Liu, Hongxia Zhu, Wei Zhang, Shuang Yan, Lanping Quan, Jinfeng Bai, Ningzhi Xu
Affiliations

Aurora kinase A induces miR-17-92 cluster through regulation of E2F1 transcription factor

Shun He et al. Cell Mol Life Sci. 2010 Jun.

Abstract

Aurora kinase A (AURKA) is an essential mitotic serine/threonine kinase and its abnormal expression is observed in many malignancies, yet the exact role for AURKA in tumorigenesis still remains elusive. Here, through a transcription factor array, we show that the transcription activity of E2F1 was increased by AURKA overexpression. Meanwhile, the E2F1 protein level was found to be upregulated and a correlation between AURKA and E2F1 expression was observed in cancer specimens. Further analysis revealed that AURKA increased E2F1 protein stability by inhibiting proteasome-dependent degradation of this protein. Additionally, a microRNA cluster, miR-17-92, was found to be upregulated upon AURKA overexpression, and this stimulation was largely repressed by E2F1 knockdown. Chromatin immunoprecipitation further demonstrated that AURKA enhanced E2F1 occupancy to the promoter of the miR-17-92 cluster. These data reveal a novel link between AURKA and microRNAs via the regulation of E2F1, providing new clues for understanding the role of AURKA in tumorigenesis.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Transcription factor activity profiling of 293TREX cells with high and low AURKA expression. a Immunoblotting analysis of AURKA expression in untreated and doxycycline(DOX)-treated 293TREX–AURKA cells. Actin was also probed to act as a loading control. b Transcription factor arrays hybridized with nuclear extracts from untreated (DOX−) or DOX-treated (DOX+) 293TREX–AURKA cells. Magnified panels show the spots representing E2F1 and NFκB transcription factors that were upregulated upon AURKA overexpression. c The summary of the transcription factors with significant changes (either upregulation or downregulation ≥two-fold) in their transcription activities upon AURKA induction. d Relative transcription activities (RTA) of E2F1 in DOX− and DOX+ cells after being normalized to respective internal controls. The data were depicted as mean ± SD (n = 4)
Fig. 2
Fig. 2
AURKA increased E2F1 transcription activity. a E2F1 DNA-binding ability was examined by electrophoresis mobility shift assay (EMSA) in nuclear extracts of 293TREX–AURKA cells. The lysates of 293TREX-Vector cells were examined as well to exclude the unspecific effects of DOX. The extracts of DOX-treated 293TREX–AURKA cells were also incubated either without probes or with probes in combination with excess cold probes to indicate the specificity of the probes. Right panel shows equal amounts of nuclear extracts were used in EMSA for the two cell lines, respectively. The upper and the lower arrows indicate exogenous and endogenous AURKA, respectively. b, c Reporter gene analysis with the pGL3-6 × E2F-luciferase plasmids. 293TREX–AURKA cells treated with or without DOX (b) and EC9706 cells transfected with 50 ng Vec, 50/100/200 ng AURKA-WT, 50 ng AURKA-KD and 50 ng E2F1 expression plasmids (c) were assayed for E2F-dependent luciferase activities. Firefly/Renilla luciferase ratios were used to calculate fold induction. Vec, pcDNA4 vector; KD, the kinase-dead mutant form of AURKA. All experiments were performed in triplicate and are depicted as mean ± SD
Fig. 3
Fig. 3
AURKA upregulated E2F1 protein expression. a E2F1 mRNA and protein levels in 293TREX–AURKA cells treated with/without DOX for 24 h. b E2F1 protein levels in SK-BR-3 cells transiently transfected with AURKA plasmids (left) and in MCF7 cells treated with AURKA siRNA for 48 h (right). P4, pcDNA4 vector; WT and KD, the wild-type and kinase-dead mutant form of AURKA, respectively; GL2 siRNA was used as a negative control for AURKA siRNA. c Immunohistochemical analysis of AURKA and E2F1 expression in breast cancer samples. Representative photographs of weak and strong staining for AURKA and E2F1 are shown. The original magnification: ×100 (pictures, size bars 100 μm), and ×400 (insets, size bars 20 μm)
Fig. 4
Fig. 4
AURKA alleviated E2F1 proteasome-dependent degradation. a Lysates of 293TREX–AURKA cells treated with or without DOX were collected at the indicated time points after addition of CHX and subjected to immunoblotting. The amounts of E2F1 were quantified by densitometry and normalized to the corresponding actin expression. The degradation rate was shown by comparing the amounts of normalized E2F1 at each time point with the original levels (0 h) in DOX+ and DOX− cells, respectively. b Lysates of 293TREX–AURKA cells treated with or without DOX were collected 8 h after addition of CHX alone or in combination with MG132 and subjected to immunoblotting
Fig. 5
Fig. 5
AURKA-induced miR-17-92 cluster expression. a Real-time PCR analysis of the miR-17-92 cluster expression in SK-BR-3 cells transiently transfected with the indicated plasmids. All experiments were performed in triplicate and are expressed as mean ± SD. b Real-time PCR analysis of the miR-17-92 cluster expression in 293TREX–AURKA cells treated with E2F1 siRNA for 48 h in the absence or presence of DOX treatment. All experiments were performed in triplicate and are expressed as mean ± SD. The efficiency of E2F1 siRNA was tested by immunoblotting with GL2 siRNA as a negative control
Fig. 6
Fig. 6
AURKA-enhanced in vivo binding of E2F1 to the promoter of the miR-17-92 cluster. a Schematic representation of the promoter of the miR-17-92 cluster. The transcription start site of C13orf25 and the primer pairs (P1 and P2) flanking the putative E2F1-binding sites are indicated. b PCR analysis of E2F1 chromatin immunoprecipitates. Chromatins prepared from 293TREX–AURKA cells treated with DOX or not were subjected to immunoprecipitation with either an anti-E2F1 antibody or mouse IgG. Associated DNA was detected by PCR with two primer pairs: P1 (flanking E2F-binding site 1) and P2 (flanking E2F-binding site 2). Input DNA of the two samples were subjected to PCR to ensure that equal amounts of total DNA were used in the immunoprecipitation. A negative control primer pair flanking a genomic DNA sequence that should not be bound by E2F1 was also used to exclude the possibility that the variances are due to contamination of different amounts of genomic DNA through unspecific immunoprecipitation
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bischoff JR, Anderson L, Zhu Y, Mossie K, Ng L, Souza B, Schryver B, Flanagan P, Clairvoyant F, Ginther C, Chan CS, Novotny M, Slamon DJ, Plowman GD. A homologue of Drosophila Aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J. 1998;17:3052–3065. doi: 10.1093/emboj/17.11.3052. - DOI - PMC - PubMed
    1. Jeng YM, Peng SY, Lin CY, Hsu HC. Overexpression and amplification of Aurora-A in hepatocellular carcinoma. Clin Cancer Res. 2004;10:2065–2071. doi: 10.1158/1078-0432.CCR-1057-03. - DOI - PubMed
    1. Rojanala S, Han H, Munoz RM, Browne W, Nagle R, Von Hoff DD, Bearss DJ. The mitotic serine threonine kinase, Aurora-2, is a potential target for drug development in human pancreatic cancer. Mol Cancer Ther. 2004;3:451–457. - PubMed
    1. Tanner MM, Grenman S, Koul A, Johannsson O, Meltzer P, Pejovic T, Borg A, Isola JJ. Frequent amplification of chromosomal region 20q12–q13 in ovarian cancer. Clin Cancer Res. 2000;6:1833–1839. - PubMed
    1. Watanabe T, Imoto I, Katahira T, Hirasawa A, Ishiwata I, Emi M, Takayama M, Sato A, Inazawa J. Differentially regulated genes as putative targets of amplifications at 20q in ovarian cancers. Jpn J Cancer Res. 2002;93:1114–1122. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources