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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 correlates with miR-20b expression levels(A) Total RNA isolated from HMEC and breast cancer cell lines MCF7, ZR75-1, HCC1419, and HCC1806 was subjected to real-time RT-PCR with a primer set for miR-20b. (B) Whole cell lysates prepared from the above cell lines were subjected to Western blot analysis using antibodies against EGR1 and GAPDH. (C) HCC1806 cells were transiently transfected with either siEGR1 (siRNA targeting EGR1) or control siRNA; the levels of EGR1 mRNA and protein were determined by real-time RT-PCR (upper panel) and Western blot analysis (lower panel). (D) HCC1806 cells were transiently transfected with either siEGR1 or control siRNA; the levels of miR-20b were determined by real-time RT-PCR. The asterisk indicates p<0.05.
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Figure 2: EGR1 correlates with miR-20b expression levels(A) Total RNA isolated from HMEC and breast cancer cell lines MCF7, ZR75-1, HCC1419, and HCC1806 was subjected to real-time RT-PCR with a primer set for miR-20b. (B) Whole cell lysates prepared from the above cell lines were subjected to Western blot analysis using antibodies against EGR1 and GAPDH. (C) HCC1806 cells were transiently transfected with either siEGR1 (siRNA targeting EGR1) or control siRNA; the levels of EGR1 mRNA and protein were determined by real-time RT-PCR (upper panel) and Western blot analysis (lower panel). (D) HCC1806 cells were transiently transfected with either siEGR1 or control siRNA; the levels of miR-20b were determined by real-time RT-PCR. The asterisk indicates p<0.05.

Mentions: To explore our hypothesis, we determined the expression of EGR1 and miR-20b, as well as the contribution of EGR1 to miR-20b transcription in breast cancer cells. qRT-PCR showed an aberrant expression of miR-20b in the breast cancer cell lines examined (Fig. 2A), which correlated with EGR1 expression (Fig. 2B), with the exception of MCF7. Knockdown of EGR1 in HCC1806 cells with the use of siEGR1 (siRNA targeting EGR1) resulted in a reduction in miR-20b expression (Fig. 2C and D). This reductioxn was particularly potent at the 50 nM siEGR1 dose (Fig. 2D). Ectopic expression of Egr1 caused induction in luciferase activity in a reporter construct harboring the wild-type miR-20b promoter in a dose-dependent fashion. This EGR1 responsiveness was completely abolished in the mutant construct (Fig. 3B). EGR1 was overexpressed in HCC1806 cells (Fig. 2B), and both real-time ChIP-PCR and conventional ChIP-PCR indicated that EGR1 was functionally enriched at miR-20b promoter in HCC1806 cells compared with normal HMEC (Fig. 3C). Furthermore, EMSA assays indicated that EGR1 specifically bound to miR-20b promoter (Fig. 3D). Taken together, these results suggested that EGR1 played a crucial role in controlling miR-20b transcription. We then determined the role of miR-20b in breast carcinogenesis.


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 correlates with miR-20b expression levels(A) Total RNA isolated from HMEC and breast cancer cell lines MCF7, ZR75-1, HCC1419, and HCC1806 was subjected to real-time RT-PCR with a primer set for miR-20b. (B) Whole cell lysates prepared from the above cell lines were subjected to Western blot analysis using antibodies against EGR1 and GAPDH. (C) HCC1806 cells were transiently transfected with either siEGR1 (siRNA targeting EGR1) or control siRNA; the levels of EGR1 mRNA and protein were determined by real-time RT-PCR (upper panel) and Western blot analysis (lower panel). (D) HCC1806 cells were transiently transfected with either siEGR1 or control siRNA; the levels of miR-20b were determined by real-time RT-PCR. The asterisk indicates p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824527&req=5

Figure 2: EGR1 correlates with miR-20b expression levels(A) Total RNA isolated from HMEC and breast cancer cell lines MCF7, ZR75-1, HCC1419, and HCC1806 was subjected to real-time RT-PCR with a primer set for miR-20b. (B) Whole cell lysates prepared from the above cell lines were subjected to Western blot analysis using antibodies against EGR1 and GAPDH. (C) HCC1806 cells were transiently transfected with either siEGR1 (siRNA targeting EGR1) or control siRNA; the levels of EGR1 mRNA and protein were determined by real-time RT-PCR (upper panel) and Western blot analysis (lower panel). (D) HCC1806 cells were transiently transfected with either siEGR1 or control siRNA; the levels of miR-20b were determined by real-time RT-PCR. The asterisk indicates p<0.05.
Mentions: To explore our hypothesis, we determined the expression of EGR1 and miR-20b, as well as the contribution of EGR1 to miR-20b transcription in breast cancer cells. qRT-PCR showed an aberrant expression of miR-20b in the breast cancer cell lines examined (Fig. 2A), which correlated with EGR1 expression (Fig. 2B), with the exception of MCF7. Knockdown of EGR1 in HCC1806 cells with the use of siEGR1 (siRNA targeting EGR1) resulted in a reduction in miR-20b expression (Fig. 2C and D). This reductioxn was particularly potent at the 50 nM siEGR1 dose (Fig. 2D). Ectopic expression of Egr1 caused induction in luciferase activity in a reporter construct harboring the wild-type miR-20b promoter in a dose-dependent fashion. This EGR1 responsiveness was completely abolished in the mutant construct (Fig. 3B). EGR1 was overexpressed in HCC1806 cells (Fig. 2B), and both real-time ChIP-PCR and conventional ChIP-PCR indicated that EGR1 was functionally enriched at miR-20b promoter in HCC1806 cells compared with normal HMEC (Fig. 3C). Furthermore, EMSA assays indicated that EGR1 specifically bound to miR-20b promoter (Fig. 3D). Taken together, these results suggested that EGR1 played a crucial role in controlling miR-20b transcription. We then determined the role of miR-20b in breast carcinogenesis.

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