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.

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.

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.

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.

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.

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.

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.