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Crucial role for early growth response-1 in the transcriptional regulation of miR-20b in breast cancer.

Li D, Ilnytskyy Y, Kovalchuk A, Khachigian LM, Bronson RT, Wang B, Kovalchuk O - Oncotarget (2013)

Bottom Line: We noted significant enrichment of EGR1 at miR-20b promoter in HCC1806 cells compared with normal human mammary epithelial cells.Suppression of miR-20b significantly inhibited HCC1806 cell proliferation and migration, and led to G0/G1 and S phase arrest.Conversely, suppression of miR-20b increased PTEN and BRCA1 levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Lethbridge, Lethbridge, Canada.

ABSTRACT
Transcriptional regulation of miRNAs that control the pathogenesis of breast cancer remains largely unknown. Here, we showed that ionizing radiation, a known breast carcinogen, triggered the differential expression of miR-20b in mammary tissues. We identified several GC-rich consensus binding motifs for the zinc finger transcription factor early growth response-1 (EGR1) in miR-20b promoter. miR-20b was upregulated by IR and its upregulation correlated with EGR1 expression in the breast cancer cell line HCC1806. Therefore, we used HCC1806 cells as a model system to explore the role of EGR1 in miR-20b transcription. siRNA knockdown of EGR1 attenuated miR-20b expression. Luciferase assays showed that whereas EGR1 stimulated luciferase activity driven by the wild-type miR-20b promoter, this induction was abolished in the mutant miR-20 promoter construct. We noted significant enrichment of EGR1 at miR-20b promoter in HCC1806 cells compared with normal human mammary epithelial cells. Suppression of miR-20b significantly inhibited HCC1806 cell proliferation and migration, and led to G0/G1 and S phase arrest. In vitro RNA-pull down assays indicated that miR-20b targets numerous tumor suppressors, including PTEN and BRCA1, which were downregulated in HCC1806. Conversely, suppression of miR-20b increased PTEN and BRCA1 levels. Moreover, immunohistochemical and FISH analyses showed that the miR-20b expression correlated significantly with EGR1 levels in breast cancer tissues. Our findings thus demonstrate for the first time that EGR1 is a key player in the transcriptional control of miR-20b, and miR-20b may in turn function as an oncogene by contributing to breast tumorigenesis via tumor suppressor targeting.

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EGR1 regulates miR-20b transcription(A) The wild-type and mutant miR-20b promoter reporters used in this project. (B) HEK293 cells were transiently transfected with pGL3-WT-miR20b-Prom or pGL3-MT-miR20b-Prom and pCB6-Egr1 or pCB6; luciferase activity was detected according to the manufacturer's instruction. (C) Real-time ChIP-PCR and conventional ChIP-PCR were performed as described in “Materials and Methods”. (D) Nuclear extracts were prepared from HCC1806 cells, and EMSA was performed using ChIP-grade antibody to EGR1 according to the manufacturer's instruction. The asterisk indicates p<0.05.
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Figure 3: EGR1 regulates miR-20b transcription(A) The wild-type and mutant miR-20b promoter reporters used in this project. (B) HEK293 cells were transiently transfected with pGL3-WT-miR20b-Prom or pGL3-MT-miR20b-Prom and pCB6-Egr1 or pCB6; luciferase activity was detected according to the manufacturer's instruction. (C) Real-time ChIP-PCR and conventional ChIP-PCR were performed as described in “Materials and Methods”. (D) Nuclear extracts were prepared from HCC1806 cells, and EMSA was performed using ChIP-grade antibody to EGR1 according to the manufacturer's instruction. The asterisk indicates p<0.05.

Mentions: Our previous studies demonstrated that IR triggered a significant and sex-specific deregulation of the microRNAome, as well as altered levels of Dicer and components of the RNA-induced silencing complex in the spleen of C57BL/6 mice [23]. To understand the microRNAs that are differentially expressed in mammary gland tissues in response to IR, six-week old female Long Evans rats were exposed to different doses/energy X-ray and sacrificed at different time points after irradiation. microRNA microarray analysis showed that 96 hours after irradiation, miR-20b was significantly reduced (Fig. 1A). This result was confirmed by quantitative real-time RT-PCR (qRT-PCR, Fig. 1B). A similar response was also displayed in human mammary epithelial cells (HMEC) 96 hours post irradiation (Fig. S1A). The qRT-PCR using RNA samples from IR-exposed mammary gland tissues at different time points showed both a time- and dose-dependent expression of miR-20b (Fig. 1C). IR also triggered a rapid and transient induction of miR-20b in HMEC cells which peaked at 24 hour post-IR (Fig. 1D and Fig. S1A), and correlated with the IR-inducible EGR-1 expression (Fig. 1E, Fig. S1B and C; correlation r=0.81926 in 30 kVp/0.1 Gy group; correlation r=0.68675 in 80 kVp/2.5 Gy group), although the EGR1 mRNA was not elevated at 6 hour post-IR. In consideration of the involvement of gene copy numbers in gene expression, we determined the changes in copy number in HMEC cells as a response to IR. However, our results showed that IR did not affect the copy number of miR-20b gene (Fig. S2), indicating the involvement of other mechanisms in the control of IR-inducible miR-20b transcription. Software-based bioinformatics analysis (Promoter 2.0 Prediction Server and Genomatix) identified several putative EGR1 binding motifs present in miR-20b promoter (Fig. 3A). We therefore hypothesized that EGR1 may play a role in miR-20b transcription.


Crucial role for early growth response-1 in the transcriptional regulation of miR-20b in breast cancer.

Li D, Ilnytskyy Y, Kovalchuk A, Khachigian LM, Bronson RT, Wang B, Kovalchuk O - Oncotarget (2013)

EGR1 regulates miR-20b transcription(A) The wild-type and mutant miR-20b promoter reporters used in this project. (B) HEK293 cells were transiently transfected with pGL3-WT-miR20b-Prom or pGL3-MT-miR20b-Prom and pCB6-Egr1 or pCB6; luciferase activity was detected according to the manufacturer's instruction. (C) Real-time ChIP-PCR and conventional ChIP-PCR were performed as described in “Materials and Methods”. (D) Nuclear extracts were prepared from HCC1806 cells, and EMSA was performed using ChIP-grade antibody to EGR1 according to the manufacturer's instruction. The asterisk indicates p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: EGR1 regulates miR-20b transcription(A) The wild-type and mutant miR-20b promoter reporters used in this project. (B) HEK293 cells were transiently transfected with pGL3-WT-miR20b-Prom or pGL3-MT-miR20b-Prom and pCB6-Egr1 or pCB6; luciferase activity was detected according to the manufacturer's instruction. (C) Real-time ChIP-PCR and conventional ChIP-PCR were performed as described in “Materials and Methods”. (D) Nuclear extracts were prepared from HCC1806 cells, and EMSA was performed using ChIP-grade antibody to EGR1 according to the manufacturer's instruction. The asterisk indicates p<0.05.
Mentions: Our previous studies demonstrated that IR triggered a significant and sex-specific deregulation of the microRNAome, as well as altered levels of Dicer and components of the RNA-induced silencing complex in the spleen of C57BL/6 mice [23]. To understand the microRNAs that are differentially expressed in mammary gland tissues in response to IR, six-week old female Long Evans rats were exposed to different doses/energy X-ray and sacrificed at different time points after irradiation. microRNA microarray analysis showed that 96 hours after irradiation, miR-20b was significantly reduced (Fig. 1A). This result was confirmed by quantitative real-time RT-PCR (qRT-PCR, Fig. 1B). A similar response was also displayed in human mammary epithelial cells (HMEC) 96 hours post irradiation (Fig. S1A). The qRT-PCR using RNA samples from IR-exposed mammary gland tissues at different time points showed both a time- and dose-dependent expression of miR-20b (Fig. 1C). IR also triggered a rapid and transient induction of miR-20b in HMEC cells which peaked at 24 hour post-IR (Fig. 1D and Fig. S1A), and correlated with the IR-inducible EGR-1 expression (Fig. 1E, Fig. S1B and C; correlation r=0.81926 in 30 kVp/0.1 Gy group; correlation r=0.68675 in 80 kVp/2.5 Gy group), although the EGR1 mRNA was not elevated at 6 hour post-IR. In consideration of the involvement of gene copy numbers in gene expression, we determined the changes in copy number in HMEC cells as a response to IR. However, our results showed that IR did not affect the copy number of miR-20b gene (Fig. S2), indicating the involvement of other mechanisms in the control of IR-inducible miR-20b transcription. Software-based bioinformatics analysis (Promoter 2.0 Prediction Server and Genomatix) identified several putative EGR1 binding motifs present in miR-20b promoter (Fig. 3A). We therefore hypothesized that EGR1 may play a role in miR-20b transcription.

Bottom Line: We noted significant enrichment of EGR1 at miR-20b promoter in HCC1806 cells compared with normal human mammary epithelial cells.Suppression of miR-20b significantly inhibited HCC1806 cell proliferation and migration, and led to G0/G1 and S phase arrest.Conversely, suppression of miR-20b increased PTEN and BRCA1 levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Lethbridge, Lethbridge, Canada.

ABSTRACT
Transcriptional regulation of miRNAs that control the pathogenesis of breast cancer remains largely unknown. Here, we showed that ionizing radiation, a known breast carcinogen, triggered the differential expression of miR-20b in mammary tissues. We identified several GC-rich consensus binding motifs for the zinc finger transcription factor early growth response-1 (EGR1) in miR-20b promoter. miR-20b was upregulated by IR and its upregulation correlated with EGR1 expression in the breast cancer cell line HCC1806. Therefore, we used HCC1806 cells as a model system to explore the role of EGR1 in miR-20b transcription. siRNA knockdown of EGR1 attenuated miR-20b expression. Luciferase assays showed that whereas EGR1 stimulated luciferase activity driven by the wild-type miR-20b promoter, this induction was abolished in the mutant miR-20 promoter construct. We noted significant enrichment of EGR1 at miR-20b promoter in HCC1806 cells compared with normal human mammary epithelial cells. Suppression of miR-20b significantly inhibited HCC1806 cell proliferation and migration, and led to G0/G1 and S phase arrest. In vitro RNA-pull down assays indicated that miR-20b targets numerous tumor suppressors, including PTEN and BRCA1, which were downregulated in HCC1806. Conversely, suppression of miR-20b increased PTEN and BRCA1 levels. Moreover, immunohistochemical and FISH analyses showed that the miR-20b expression correlated significantly with EGR1 levels in breast cancer tissues. Our findings thus demonstrate for the first time that EGR1 is a key player in the transcriptional control of miR-20b, and miR-20b may in turn function as an oncogene by contributing to breast tumorigenesis via tumor suppressor targeting.

Show MeSH
Related in: MedlinePlus