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. 2009 May;37(8):2584-95.
doi: 10.1093/nar/gkp117. Epub 2009 Mar 5.

Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells

Nalinie S Wickramasinghe  1 , Tissa T Manavalan, Susan M Dougherty, Krista A Riggs, Yong Li, Carolyn M Klinge
Affiliations

Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells

Nalinie S Wickramasinghe et al. Nucleic Acids Res. 2009 May.

Abstract

Select changes in microRNA (miRNA) expression correlate with estrogen receptor alpha (ER alpha) expression in breast tumors. miR-21 is higher in ER alpha positive than negative tumors, but no one has examined how estradiol (E(2)) regulates miR-21 in breast cancer cells. Here we report that E(2) inhibits miR-21 expression in MCF-7 human breast cancer cells. The E(2)-induced reduction in miR-21 was inhibited by 4-hydroxytamoxifen (4-OHT), ICI 182 780 (Faslodex), and siRNA ER alpha indicating that the suppression is ER alpha-mediated. ER alpha and ER beta agonists PPT and DPN inhibited and 4-OHT increased miR-21 expression. E(2) increased luciferase activity from reporters containing the miR-21 recognition elements from the 3'-UTRs of miR-21 target genes, corroborating that E(2) represses miR-21 expression resulting in a loss of target gene suppression. The E(2)-mediated decrease in miR-21 correlated with increased protein expression of endogenous miR-21-targets Pdcd4, PTEN and Bcl-2. siRNA knockdown of ER alpha blocked the E(2)-induced increase in Pdcd4, PTEN and Bcl-2. Transfection of MCF-7 cells with antisense (AS) to miR-21 mimicked the E(2)-induced increase in Pdcd4, PTEN and Bcl-2. These results are the first to demonstrate that E(2) represses the expression of an oncogenic miRNA, miR-21, by activating estrogen receptor in MCF-7 cells.

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Figures

Figure 1.
Figure 1.
E2 inhibits miR-21 expression. Summary of Q-PCR data on (mature) miR-21 expression. MCF-7 cells were treated with EtOH, 10 nM E2, 10 nM PPT (ERα-selective), or 10 nM DPN (ERβ-selective) for 6 h. as indicated by the different fills. Where indicated MCF-7 cells were pretreated with 100 nM ICI 182 780 [ICI, an ER antagonist termed a ‘selective ER disrupter’ (SERD)] or 100 nM 4-OHT for 6 h and then ethanol or 10 nM E2 was added for an additional 6 h. Values are fold increase compared to EtOH for each miRNA and were calculated as described in ‘Materials and Methods’ section. Values are the average of three to eight separate experiments ± SEM. *Significantly different from the EtOH control, P < 0.05. **Significantly different from E2, P < 0.05.
Figure 2.
Figure 2.
Luciferase reporter assay of putative miR-21 target genes and the effect of antisense (AS) to miR-21 on reporter activity. (A) Model of transient transfection assays in MCF-7 cells. MCF-7 cells were transiently transfected with pGL3-pro-luciferase and pRL-TK parental or pRL-TK containing putative miR-21 MREs from target genes (Supplementary Table 1) cloned in the 3′UTR as described in ‘Materials and methods’ section. Expected results are indicated without E2 (A) and when cells are treated with E2 (B). (C) MCF-7 cells were transfected as indicated and treated with EtOH or 10 nM E2 for 24 h. Renilla luciferase was normalized by firefly luciferase to correct for transfection efficiency. Values are the average ± SEM of triplicate determinations. *Significantly different from EtOH control, P < 0.01. (D) MCF-7 cells were transfected with 2′-O-Me-antisense-miR-21 (ASmiR-21). Renilla luciferase reporter gene expression from the indicated gene MREs was determined and data analyzed as described in ‘Materials and Methods’ section. The control was a random-sequence 2′-O-Me modified RNA (control AS) as described in Materials and methods section. Values are the average ± SEM of triplicate determinations. *Significantly different from control AS, P < 0.05. (E) MCF-7 cells were transfected with the pRL-tk-MREs or FL 3′-UTRs as indicated. Indicated cells were co-transfected with ASmiR-21 or a control AS. Cells were treated with EtOH or 10 nM E2 as indicated for 24 h. Dual luciferase reporter assays were performed and data quantitated as described in ‘Materials and Methods’ section. Values are the average ± SEM of triplicate determinations normalized to EtOH for each construct except that cells transfected with the ASmiR-21 were normalized against the control AS-EtOH value. aSignificantly different from EtOH control, P < 0.01. bSignificantly different from control AS transfected values, P < 0.01.
Figure 3.
Figure 3.
Regulation of miR-21 transcription in MCF-7 cells. (A) Effects of E2, 4-OHT and ICI 182 780 (ICI) on the primary miR-21 (pri-miR-21) gene promoter in the sense (pmiR-21s-luc) or antisense (as) pmiR-21as-luc orientation. MCF-7 cells were transfected with pri-miR-21s-luc or pri-miR-21as-luc (hatched bars, values were very low) (45) and Renilla luciferase as an internal control. Cells were treated with the indicated concentrations of E2, 4-OHT, or ICI for 24 h. Dual luciferase assays were performed and luciferase values were divided by Renilla values in the same sample. Values are the average ± SEM of triplicate determinations normalized to EtOH for the pmiR-21s-luc construct. *Significantly different from EtOH control, P < 0.05. **Significantly different from 4-OHT alone, P < 0.05. ***Significantly different from 10 nM E2, P < 0.05. (B) E2 does not affect TMEM49 transcription in MCF-7 cells. miR-21 is encoded within the 10th intron of the TMEM49 gene (56). MCF-7 cells were treated with EtOH or 10 nM E2 for 6 h, total RNA was reverse transcribed and Q–PCR was performed. TMEM49 was normalized to 18S. Values are the average ± SEM of triplicate determinations normalized to EtOH. (C) The E2-induced decrease in miR-21 expression in MCF-7 cells is mediated in a primary transcriptional/genomic and secondary estrogen-target-dependent manner. MCF-7 cells were pre-treated with stripped medium or stripped medium containing 10 μg/ml ActD or CHX for 1 h before treatment with vehicle control (EtOH), or 10 nM E2 for 6 h as described in ‘Materials and Methods’ section. miR-21 expression was determined using Q-PCR as described in ‘Materials and Methods’ section. The bar graph summarizes the fold change in miR-21 expression relative to no pretreatment (No pretx)-EtOH-treated cells.
Figure 4.
Figure 4.
Effect of ER ligands on endogenous miR21 target gene mRNA and protein expression in MCF-7 cells. MCF-7 cells were serum-starved for 48 h and then treated with EtOH, 10 nM E2, 10 nM PPT (ERα selective), or 10 nM DPN (ERβ selective) for 6 h prior to RNA isolation (A) or 24 h prior to WCE preparation (B) as described in ‘Materials and methods’ section. (A) Q-PCR was performed for the indicated genes and fold-expression determined compared to EtOH as described in ‘Materials and Methods’ section. Values are the average of four separate determinations ± SEM. (B) Western blot for the indicated proteins. The membrane was stripped and reprobed for β-actin for normalization as described in ‘Materials and Methods’ section. The blot shown is representative of three separate biological replicates. (C) Western data are presented as relative to non-treated (No TX) MCF-7 cells. The values in C are the mean ± SEM of three separate experiments. *Significantly different from the EtOH value for each protein, P < 0.01.
Figure 5.
Figure 5.
AS-miR-21 increases the expression of PTEN, PDCD4 and Bcl-2. (A) The specificity of AS-miR-21 to decrease miR-21was examined by Q-PCR in parallel with miR-125a and miR-30b as negative controls. MCF-7 cells were not transfected (unTF) or were transfected with AS-control or AS-miR-21 for 48 h prior to RNA harvest. Q-PCR was performed for the indicated miRs. The values are the average of three separate experiments, each run in triplicate, ± SD. (B) MCF-7 cells were transfected with AS-control or AS-miR-21 for 24 h prior to serum deprivation for 48 h and then 24 h treatment with EtOH, 10 nM E2, PPT or DPN, as indicated. WCE were used for western blot for the indicated proteins as described in ‘Materials and methods’ section. The membrane was stripped and reprobed for β-actin for normalization as described in Materials and Methods section. The blot shown is representative of three separate biological replicates. (C) The values graphed are the mean ± SEM of the normalized western data (each protein was normalized to β-actin input and then the ratio of each protein/β-actin in the AS-control in EtOH-treated samples was set to one in each experiment) in three separate experiments. *Significantly different from the EtOH AS-control for each protein, P < 0.05.
Figure 6.
Figure 6.
ERα, but not ERβ, knockdown inhibits the E2-mediated decrease in miR-21 and thus reverses miR-21 target gene expression. (A) MCF-7 cells were not transfected (Not TF) or transfected with siControl RNA or siERα as described in ‘Materials and Methods’ section for 48 h and WCE were analyzed for ERα and ERβ by western blot as described in ‘Materials and Methods’ section. The same membrane was stripped and reprobed for β-actin for normalization. The% ERα knockdown was calculated relative to the Not TF control. (B) MCF-7 cells were transfected with siControl RNA or siERα for 48 h prior to treatment with EtOH or 10 nM E2, PPT, or DPN for 6 h. RNA and protein were extracted and Q-PCR (B and C) or western blots (D and E) were performed for the indicated miR-21 targets as described in ‘Materials and Methods’ section. The blots shown are representative of three separate biological replicates. The values in (E) are the mean ± SEM of three to four separate experiments. (F) MCF-7 cells were transfected with siControl RNA or siERβ for 48 h prior to treatment with EtOH or 10 nM E2 for 6 h. MiR-21 expression is the mean fold change ± SEM of four samples. Values are mean ± SEM. *Significantly different from the EtOH siControl for each protein, P < 0.05. **Significantly different from E2, PPT or DPN siControl value for that protein, P < 0.05.
Figure 7.
Figure 7.
ER regulates miR-21 expression and its downstream targets in a ligand-dependent manner. E2-ER (ERα and/or ERβ) inhibits miR-21 expression resulting in a loss of repression (indicated by the Xs) of Pdcd4, PTEN and Bcl-2 protein expression. E2-ERα directly increases BCL2 transcription (arrow, +). 4-OHT and ICI block ER-induced inhibition of miR-21 expression. E2-ER also regulates the expression of other miRNAs and mRNAs that, in turn, regulate other cellular pathways which impact the expression of PDCD4, PTEN and BCL2.
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