Estradiol-regulated microRNAs control estradiol response in breast cancer cells - PubMed (original) (raw)

Estradiol-regulated microRNAs control estradiol response in breast cancer cells

Poornima Bhat-Nakshatri et al. Nucleic Acids Res. 2009 Aug.

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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Figures

Figure 1.

Figure 1.

(A) E2-inducible expression of Let-7f, miR98 and miR-21 in MCF-7p and MCF-7AKT cells. MCF-7p and MCF-7AKT cells were treated with E2 (10−8M) for indicated time and microRNA was subjected qRT–PCR. RNU66, small RNAs encoded in the intron of RPL5 gene (chr1:93 018 360–93 018 429) was used for normalization between samples. Mean plus standard error of the mean (SEM) are shown. *P < 0.03, ethanol versus 1-h E2-treated cells; **P < 0.001 ethanol versus 4 h E2-treated cells. (B) The effect of E2 on the expression of Let-7f, miR-98 and miR-21 in MCF-7 (top), T47-D (middle) and BT-474 (bottom) cells. (C) The effect of fulvestrant treatment on basal and E2-regulated expression of Let-7f, miR-98 and miR-21. Cells were pretreated with 100 nM fulvestrant for 24 h and then treated with E2 for 4 h. *P < 0.05, ethanol versus E2-treated; **P < 0.05, ethanol versus fulvestrant treated cells.

Figure 2.

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.

Figure 3.

Figure 3.

The regulatory regions of miR-21 contain ERα-binding sites. (A) ERα DNA-binding patterns to the genomic region harboring miR-21 gene on chromosome 17. Black bars represent ERα-binding sites in two cell types observed under ethanol and E2-treated condition. (B) ChIP analysis of ERα binding to the genomic region (central bar) shown in A. ChIP DNA obtained with ERα antibody was subjected to semi-quantitative PCR (top) or Q-PCR (bottom) with specific primers and relative enrichment of ERα binding is shown after normalizing ERα binding in ethanol MCF-7p or MCF-7AKT cells to one unit. The level of ERα binding in E2-treated MCF-7AKT cells was significantly higher than in untreated cells.

Figure 4.

Figure 4.

Dicer is an E2-inducible gene. (A) ERα DNA-binding patterns to the genomic region harboring Dicer. Black bars represent ERα-binding sites in two cell types observed under ethanol-treated or E2-treated condition. (B) E2-inducible expression of Dicer in MCF-7p and MCF-7AKT cells was measured by northern blotting (top panel). The same blot was reprobed with 36B4 to ensure integrity of RNA. qRT–PCR confirming E2-inducible expression of Dicer is also shown (mean plus SEM, bottom panel). *P < 0.04 ethanol-treated versus E2-treated cells.

Figure 5.

Figure 5.

A model depicting the possible effects of E2-regulated microRNAs on E2 response and differentiated phenotype of ERα-positive breast cancer cells.

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