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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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PTEN and BRCA1 are direct targets of miR-20b(A) The network of the predicted targets of hsa-miR-20b was generated using STRING 9.0. (B) Diagram of 3'UTR sequences of PTEN and BRCA1 targeted by hsa-miR-20b. (C) Whole cellular lysates prepared from HMEC, HCC1806, and HCC1806 transfected with either 50 nM miR-20b inhibitor or non-specific control for 72 hours were subjected to Western blot analysis using antibodies specific to PTEN and BRCA1. (D) HEK293 cells grown to 90% confluency were cotransfected with either pGL3-PTEN or pGL3-BRCA1 reporter, and the indicated concentration of hsa-miR-20b or 50 nM nonspecific miRNA as a control; 24 hours after transfection, luciferase activity was detected using Dual-Luciferase Reporter Assay System according to the manufacturer's instruction. The asterisk indicates p<0.05.
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Figure 5: PTEN and BRCA1 are direct targets of miR-20b(A) The network of the predicted targets of hsa-miR-20b was generated using STRING 9.0. (B) Diagram of 3'UTR sequences of PTEN and BRCA1 targeted by hsa-miR-20b. (C) Whole cellular lysates prepared from HMEC, HCC1806, and HCC1806 transfected with either 50 nM miR-20b inhibitor or non-specific control for 72 hours were subjected to Western blot analysis using antibodies specific to PTEN and BRCA1. (D) HEK293 cells grown to 90% confluency were cotransfected with either pGL3-PTEN or pGL3-BRCA1 reporter, and the indicated concentration of hsa-miR-20b or 50 nM nonspecific miRNA as a control; 24 hours after transfection, luciferase activity was detected using Dual-Luciferase Reporter Assay System according to the manufacturer's instruction. The asterisk indicates p<0.05.

Mentions: Because of the upregulation of miR-20b in HCC1806 cells, we selected this cell line as a model system to functionally suppress miR-20b with the use of specific inhibitors. HCC1806 cell proliferation was significantly suppressed by miR-20b inhibitor in an MTT assay (Fig. 4A), and HCC1806 cell migration was likewise inhibited in a wound healing assay (Fig. 4B and C). Inhibition of miR-20b also interestingly resulted in G0/G1 and S phase cell cycle arrest (Fig. 4D), although miR-20b inhibitor did not affect apoptosis (Fig. S3). To identify the target molecules of miR-20b that may be involved in these pathological processes, molecules that bind to miR-20b were pulled down in vitro and subjected to deep sequencing analysis. Software predictions by MIRANDA and RNAhybrid showed that miR-20b could bind to the 3' UTRs of many tumor suppressors (Fig. 5A) that are primarily associated with cell proliferation, invasion, apoptosis, and cell cycle control. Among the predicted targets of miR-20b, phosphatase and tensin homolog (PTEN, Fig. 5B) and breast cancer 1 gene (BRCA1, Fig. 5B) are critical in the maintenance of genomic stability, negative regulation of proliferative signaling, and prevention of cancer. Western blot analysis showed that PTEN and BRCA1 were downregulated in HCC1806 cells compared with HMEC, and that were inversely correlated with miR-20b expression in these cell lines (Fig. 5C, Fig. 2A). By contrast, suppression of miR-20b resulted in an elevation of the aforementioned proteins in HCC1806 cells (Fig. 5C). Luciferase activity in both pGL3-PTEN and pGL3-BRCA1 reporters was significantly reduced by miR-20b (Fig. 5D), suggesting that PTEN and BRCA1 were direct targets of miR-20b.


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)

PTEN and BRCA1 are direct targets of miR-20b(A) The network of the predicted targets of hsa-miR-20b was generated using STRING 9.0. (B) Diagram of 3'UTR sequences of PTEN and BRCA1 targeted by hsa-miR-20b. (C) Whole cellular lysates prepared from HMEC, HCC1806, and HCC1806 transfected with either 50 nM miR-20b inhibitor or non-specific control for 72 hours were subjected to Western blot analysis using antibodies specific to PTEN and BRCA1. (D) HEK293 cells grown to 90% confluency were cotransfected with either pGL3-PTEN or pGL3-BRCA1 reporter, and the indicated concentration of hsa-miR-20b or 50 nM nonspecific miRNA as a control; 24 hours after transfection, luciferase activity was detected using Dual-Luciferase Reporter Assay System according to the manufacturer's instruction. The asterisk indicates p<0.05.
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Related In: Results  -  Collection

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Show All Figures
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Figure 5: PTEN and BRCA1 are direct targets of miR-20b(A) The network of the predicted targets of hsa-miR-20b was generated using STRING 9.0. (B) Diagram of 3'UTR sequences of PTEN and BRCA1 targeted by hsa-miR-20b. (C) Whole cellular lysates prepared from HMEC, HCC1806, and HCC1806 transfected with either 50 nM miR-20b inhibitor or non-specific control for 72 hours were subjected to Western blot analysis using antibodies specific to PTEN and BRCA1. (D) HEK293 cells grown to 90% confluency were cotransfected with either pGL3-PTEN or pGL3-BRCA1 reporter, and the indicated concentration of hsa-miR-20b or 50 nM nonspecific miRNA as a control; 24 hours after transfection, luciferase activity was detected using Dual-Luciferase Reporter Assay System according to the manufacturer's instruction. The asterisk indicates p<0.05.
Mentions: Because of the upregulation of miR-20b in HCC1806 cells, we selected this cell line as a model system to functionally suppress miR-20b with the use of specific inhibitors. HCC1806 cell proliferation was significantly suppressed by miR-20b inhibitor in an MTT assay (Fig. 4A), and HCC1806 cell migration was likewise inhibited in a wound healing assay (Fig. 4B and C). Inhibition of miR-20b also interestingly resulted in G0/G1 and S phase cell cycle arrest (Fig. 4D), although miR-20b inhibitor did not affect apoptosis (Fig. S3). To identify the target molecules of miR-20b that may be involved in these pathological processes, molecules that bind to miR-20b were pulled down in vitro and subjected to deep sequencing analysis. Software predictions by MIRANDA and RNAhybrid showed that miR-20b could bind to the 3' UTRs of many tumor suppressors (Fig. 5A) that are primarily associated with cell proliferation, invasion, apoptosis, and cell cycle control. Among the predicted targets of miR-20b, phosphatase and tensin homolog (PTEN, Fig. 5B) and breast cancer 1 gene (BRCA1, Fig. 5B) are critical in the maintenance of genomic stability, negative regulation of proliferative signaling, and prevention of cancer. Western blot analysis showed that PTEN and BRCA1 were downregulated in HCC1806 cells compared with HMEC, and that were inversely correlated with miR-20b expression in these cell lines (Fig. 5C, Fig. 2A). By contrast, suppression of miR-20b resulted in an elevation of the aforementioned proteins in HCC1806 cells (Fig. 5C). Luciferase activity in both pGL3-PTEN and pGL3-BRCA1 reporters was significantly reduced by miR-20b (Fig. 5D), suggesting that PTEN and BRCA1 were direct targets of miR-20b.

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