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Estradiol-regulated microRNAs control estradiol response in breast cancer cells.

Bhat-Nakshatri P, Wang G, Collins NR, Thomson MJ, Geistlinger TR, Carroll JS, Brown M, Hammond S, Srour EF, Liu Y, Nakshatri H - Nucleic Acids Res. (2009)

Bottom Line: E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins.Several E2-regulated microRNA genes are associated with ERalpha-binding sites or located in the intragenic region of estrogen-regulated genes.Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.

ABSTRACT
Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). E2-inducible proteins c-Myc and E2Fs are required for optimal ERalpha activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERalpha-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERalpha-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.

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Related in: MedlinePlus

Putative targets of E2-induced microRNAs. (A) MCF-7p and MCF-7AKT cells were treated with E2 for indicated time and western blotting was performed. (B) 3′-UTR of AIB1 or c-Myc reduces E2-inducible expression of a luciferase reporter under the control of CMV enhancer-promoter. MCF-7 cells were transfected with indicated reporters along with the internal control Renila-luciferase under TK promoter-enhancer and luciferase activity was measured in untreated and E2-treated cells. *P < 0.05, ethanol treated versus E2-treated cells. (C) LNA against Let-7f/miR-98 and miR-21 differentially affect basal and E2-inducible levels of c-Myc and E2F2 proteins in MCF-7p cells. Cells were treated with control or specific LNA and treated with E2 for 24 h. Western blot analysis was done with indicated antibodies. (D) Expression levels of E2F-1, E2F-2, c-Myc and AIB1 from three to five independent experiments, as measured by densitometric scanning (Mean plus SEM), are indicated. *P < 0.05, ethanol versus E2-treated cells; **P < 0.05, control LNA treated versus Let-7f/mir-98 or miR-21 LNA-treated cells. Note the effects of LNA against Let-7f/miR98 and miR-21 on E2F2 and c-Myc but not on E2F1 and AIB1 protein levels.
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Figure 2: Putative targets of E2-induced microRNAs. (A) MCF-7p and MCF-7AKT cells were treated with E2 for indicated time and western blotting was performed. (B) 3′-UTR of AIB1 or c-Myc reduces E2-inducible expression of a luciferase reporter under the control of CMV enhancer-promoter. MCF-7 cells were transfected with indicated reporters along with the internal control Renila-luciferase under TK promoter-enhancer and luciferase activity was measured in untreated and E2-treated cells. *P < 0.05, ethanol treated versus E2-treated cells. (C) LNA against Let-7f/miR-98 and miR-21 differentially affect basal and E2-inducible levels of c-Myc and E2F2 proteins in MCF-7p cells. Cells were treated with control or specific LNA and treated with E2 for 24 h. Western blot analysis was done with indicated antibodies. (D) Expression levels of E2F-1, E2F-2, c-Myc and AIB1 from three to five independent experiments, as measured by densitometric scanning (Mean plus SEM), are indicated. *P < 0.05, ethanol versus E2-treated cells; **P < 0.05, control LNA treated versus Let-7f/mir-98 or miR-21 LNA-treated cells. Note the effects of LNA against Let-7f/miR98 and miR-21 on E2F2 and c-Myc but not on E2F1 and AIB1 protein levels.

Mentions: We focused our studies only to those genes that are involved in E2-signaling pathways and/or regulated by E2 at the transcriptional level and additionally controlled at the post-transcriptional level by E2-regulated microRNAs as part of an autoregulatory loop (Figure 2A). We observed repression of AIB1 and induction of EZH2 by E2 in both cell types. Additionally, while E2F1 and E2F2 displayed a delayed E2-inducible increase in protein levels in MCF-7p cells, basal levels of both of these proteins were elevated with a further E2-inducible increase in MCF-7AKT cells. Elevated basal levels of these two proteins in MCF-7AKT cells correlate with a lower level of miR20b in these cells compared to MCF-7p cells (Table 1). Previously, we reported little difference in the kinetics of E2-inducible expression of c-Myc mRNA in parental and CA-AKT overexpressing MCF-7 cells (3). However, at the protein level, E2-mediated increase in c-Myc is advanced in MCF-7AKT cells compared to MCF-7p cells (Figure 2A). None of the previous microarray profiling studies including our study observed an effect of E2 on EZH2 mRNA level and our ChIP-chip data did not identify ERα-binding sites associated with the EZH2 gene. Furthermore, RT–PCR analysis has confirmed the lack of an E2 effect on the mRNA levels of EZH2 (data not shown). Thus, E2 may control translation of AIB1 and EZH2 through microRNAs.


Estradiol-regulated microRNAs control estradiol response in breast cancer cells.

Bhat-Nakshatri P, Wang G, Collins NR, Thomson MJ, Geistlinger TR, Carroll JS, Brown M, Hammond S, Srour EF, Liu Y, Nakshatri H - Nucleic Acids Res. (2009)

Putative targets of E2-induced microRNAs. (A) MCF-7p and MCF-7AKT cells were treated with E2 for indicated time and western blotting was performed. (B) 3′-UTR of AIB1 or c-Myc reduces E2-inducible expression of a luciferase reporter under the control of CMV enhancer-promoter. MCF-7 cells were transfected with indicated reporters along with the internal control Renila-luciferase under TK promoter-enhancer and luciferase activity was measured in untreated and E2-treated cells. *P < 0.05, ethanol treated versus E2-treated cells. (C) LNA against Let-7f/miR-98 and miR-21 differentially affect basal and E2-inducible levels of c-Myc and E2F2 proteins in MCF-7p cells. Cells were treated with control or specific LNA and treated with E2 for 24 h. Western blot analysis was done with indicated antibodies. (D) Expression levels of E2F-1, E2F-2, c-Myc and AIB1 from three to five independent experiments, as measured by densitometric scanning (Mean plus SEM), are indicated. *P < 0.05, ethanol versus E2-treated cells; **P < 0.05, control LNA treated versus Let-7f/mir-98 or miR-21 LNA-treated cells. Note the effects of LNA against Let-7f/miR98 and miR-21 on E2F2 and c-Myc but not on E2F1 and AIB1 protein levels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Putative targets of E2-induced microRNAs. (A) MCF-7p and MCF-7AKT cells were treated with E2 for indicated time and western blotting was performed. (B) 3′-UTR of AIB1 or c-Myc reduces E2-inducible expression of a luciferase reporter under the control of CMV enhancer-promoter. MCF-7 cells were transfected with indicated reporters along with the internal control Renila-luciferase under TK promoter-enhancer and luciferase activity was measured in untreated and E2-treated cells. *P < 0.05, ethanol treated versus E2-treated cells. (C) LNA against Let-7f/miR-98 and miR-21 differentially affect basal and E2-inducible levels of c-Myc and E2F2 proteins in MCF-7p cells. Cells were treated with control or specific LNA and treated with E2 for 24 h. Western blot analysis was done with indicated antibodies. (D) Expression levels of E2F-1, E2F-2, c-Myc and AIB1 from three to five independent experiments, as measured by densitometric scanning (Mean plus SEM), are indicated. *P < 0.05, ethanol versus E2-treated cells; **P < 0.05, control LNA treated versus Let-7f/mir-98 or miR-21 LNA-treated cells. Note the effects of LNA against Let-7f/miR98 and miR-21 on E2F2 and c-Myc but not on E2F1 and AIB1 protein levels.
Mentions: We focused our studies only to those genes that are involved in E2-signaling pathways and/or regulated by E2 at the transcriptional level and additionally controlled at the post-transcriptional level by E2-regulated microRNAs as part of an autoregulatory loop (Figure 2A). We observed repression of AIB1 and induction of EZH2 by E2 in both cell types. Additionally, while E2F1 and E2F2 displayed a delayed E2-inducible increase in protein levels in MCF-7p cells, basal levels of both of these proteins were elevated with a further E2-inducible increase in MCF-7AKT cells. Elevated basal levels of these two proteins in MCF-7AKT cells correlate with a lower level of miR20b in these cells compared to MCF-7p cells (Table 1). Previously, we reported little difference in the kinetics of E2-inducible expression of c-Myc mRNA in parental and CA-AKT overexpressing MCF-7 cells (3). However, at the protein level, E2-mediated increase in c-Myc is advanced in MCF-7AKT cells compared to MCF-7p cells (Figure 2A). None of the previous microarray profiling studies including our study observed an effect of E2 on EZH2 mRNA level and our ChIP-chip data did not identify ERα-binding sites associated with the EZH2 gene. Furthermore, RT–PCR analysis has confirmed the lack of an E2 effect on the mRNA levels of EZH2 (data not shown). Thus, E2 may control translation of AIB1 and EZH2 through microRNAs.

Bottom Line: E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins.Several E2-regulated microRNA genes are associated with ERalpha-binding sites or located in the intragenic region of estrogen-regulated genes.Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.

ABSTRACT
Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). E2-inducible proteins c-Myc and E2Fs are required for optimal ERalpha activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERalpha-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERalpha-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.

Show MeSH
Related in: MedlinePlus