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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 expression is correlated with miR-20b expression in breast cancer tissues(A) Representatives of EGR1 and hsa-miR-20b stainings in the same sections of breast cancer tissue arrays. (B) Statistical and correlation analyses of EGR1 and hsa-miR-20b expression in breast cancer tissues. (C) Representatives of EGR1 and hsa-miR-20b staining in the same sections of metastatic breast cancer tissue arrays. (D) Statistical and correlation analyses of EGR1 and hsa-miR-20b expression in metastatic breast cancer tissues.
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Figure 6: EGR1 expression is correlated with miR-20b expression in breast cancer tissues(A) Representatives of EGR1 and hsa-miR-20b stainings in the same sections of breast cancer tissue arrays. (B) Statistical and correlation analyses of EGR1 and hsa-miR-20b expression in breast cancer tissues. (C) Representatives of EGR1 and hsa-miR-20b staining in the same sections of metastatic breast cancer tissue arrays. (D) Statistical and correlation analyses of EGR1 and hsa-miR-20b expression in metastatic breast cancer tissues.

Mentions: To further confirm the role of EGR1 in miR-20b transcription in diseased tissues, immunohistochemical staining and FISH analysis were performed to determine the expression of EGR1 and miR-20b in breast cancer tissue arrays. EGR1 was upregulated in 40% breast cancer tissues, and miR-20b was elevated in 30% breast cancer tissues examined: upregulated EGR1 correlated significantly with upregulated miR-20b in the normal, benign, and tumor tissues tested (Fig. 6A and B; r=0.99, p=0.032). Likewise, downregulation of EGR1 also correlated with downregulated miR-20b in the normal, benign, and tumor tissues examined (Fig. 6A and B; r=0.99, p=0.054). More important, EGR1 was overexpressed in 50% of the metastatic breast cancer tissues examined. Once again, this overexpresion correlated strongly with an upregulation of miR-20b (Fig. 6C and D). These results further confirm the role of EGR1 in the transcriptional control of miR-20b and that aberrant expression contributed to the development of breast cancer, particularly metastatic breast cancer.


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 expression is correlated with miR-20b expression in breast cancer tissues(A) Representatives of EGR1 and hsa-miR-20b stainings in the same sections of breast cancer tissue arrays. (B) Statistical and correlation analyses of EGR1 and hsa-miR-20b expression in breast cancer tissues. (C) Representatives of EGR1 and hsa-miR-20b staining in the same sections of metastatic breast cancer tissue arrays. (D) Statistical and correlation analyses of EGR1 and hsa-miR-20b expression in metastatic breast cancer tissues.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: EGR1 expression is correlated with miR-20b expression in breast cancer tissues(A) Representatives of EGR1 and hsa-miR-20b stainings in the same sections of breast cancer tissue arrays. (B) Statistical and correlation analyses of EGR1 and hsa-miR-20b expression in breast cancer tissues. (C) Representatives of EGR1 and hsa-miR-20b staining in the same sections of metastatic breast cancer tissue arrays. (D) Statistical and correlation analyses of EGR1 and hsa-miR-20b expression in metastatic breast cancer tissues.
Mentions: To further confirm the role of EGR1 in miR-20b transcription in diseased tissues, immunohistochemical staining and FISH analysis were performed to determine the expression of EGR1 and miR-20b in breast cancer tissue arrays. EGR1 was upregulated in 40% breast cancer tissues, and miR-20b was elevated in 30% breast cancer tissues examined: upregulated EGR1 correlated significantly with upregulated miR-20b in the normal, benign, and tumor tissues tested (Fig. 6A and B; r=0.99, p=0.032). Likewise, downregulation of EGR1 also correlated with downregulated miR-20b in the normal, benign, and tumor tissues examined (Fig. 6A and B; r=0.99, p=0.054). More important, EGR1 was overexpressed in 50% of the metastatic breast cancer tissues examined. Once again, this overexpresion correlated strongly with an upregulation of miR-20b (Fig. 6C and D). These results further confirm the role of EGR1 in the transcriptional control of miR-20b and that aberrant expression contributed to the development of breast cancer, particularly metastatic breast cancer.

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