Differential expression of microRNA expression in tamoxifen-sensitive MCF-7 versus tamoxifen-resistant LY2 human breast cancer cells

Abstract

Microarrays identified miRNAs differentially expressed and 4-hydroxytamoxifen (4-OHT) regulated in MCF-7 endocrine-sensitive versus resistant LY2 human breast cancer cells. 97 miRNAs were differentially expressed in MCF-7 versus LY2 cells. Opposite expression of miRs-10a, 21, 22, 29a, 93, 125b, 181, 200a, 200b, 200c, 205, and 222 was confirmed. Bioinformatic analyses to impute the biological significance of these miRNAs identified 36 predicted gene targets from those regulated by 4-OHT in MCF-7 cells. Agreement in the direction of anticipated regulation was detected for 12 putative targets. These miRNAs with opposite expression between the two cell lines may be involved in endocrine resistance.

Figure 1. Heat map (hierarchical clusters) of significant differences in miRNA expression between MCF-7 and LY2 cells

MCF-7 and LY2 cells were treated with EtOH, 10 nM E₂ or 100 nM 4-OHT for 6 h and miRNA expression determined by microarray analyses of 4 separate experiments. The heatmap shows 97 miRNAs significantly differentially expressed (adjusted p-value < 0.10) between cell lines (MCF-7/EtOH versus LY2/EtOH and MCF-7/4-OHT versus LY2/4-OHT, see Tables 1 and 2, respectively). Each row of the heat map represents a gene, and each column represents a cell line/treatment group (as labeled at the bottom. Yellow indicates an increase in miRNA gene expression (relative to the other expression measurements in the same row) and orange/red indicates a decrease in expression.

Figure 2. Venn diagrams summarizing differentially expressed (DE) miRNAs

A) Venn diagram of miRNAs differentially expressed between TAM-sensitive (MCF-7) and TAM-resistant (LY2) human breast cancer cells by the indicated treatments (EtOH and 4-OHT). Cells were treated as described in Figure 1. B) Venn diagram showing miRNAs either UP- (increased expression ) or DOWN- (reduced expression) regulated between TAM-sensitive (MCF-7) and TAM-resistant (LY2) human breast cancer cells by treatment.

Figure 3. Selected miRNAs are differentially expressed in MCF-7 (TAM-S) and LY2 (TAM-R) breast cancer cells

These 12 miRNAs were identified as differentially expressed in microarray analysis of miRNAs in EtOH, E₂ or 4-OHT treated cells. Values are log2(Hy3/Hy5) ratios in the sample versus the common reference pool. Each value is the avg. ± SEM of 4 separate experiments.

Figure 4. Selection of endogenous control genes for analysis of miRNA expression by Q-PCR

The expression of RNU6-1 (U6) and RNU48 (U48) RNA genes, traditionally used as controls for miRNA expression and eight candidate endogenous control genes (ECG) identified by miRNA microarray: high signal: miR-16, Let-7f, and 5SrRNA; medium signal: SNORD38D (U38B), Let-7d, and miR-340; low signal: miR-765 and miR-744 were examined in MCF-7 (A) and LY2 (B) cells treated for 6 h with EtOH (vehicle control) or 100 nM 4-OHT. C) The expression of the Let-7 family members was determined Exiqon Microarray analysis of miRNAs in EtOH, E₂ or 4-OHT treated cells, as indicated. Values are log2(Hy3/Hy5) ratios in the sample versus the common reference pool. Each value is the avg. ± SEM of 4 separate experiments. D) The relative expression of MYC in MCF-7 and LY2 cells treated with EtOH, 10 nM E₂, or 100 nM 4-OHT for 6 h was determined by Q-PCR and CT values are the mean of 3 separate determinations ± SEM. E) Relative MYC expression in MCF-7 was normalized to 18S. * p < 0.05 versus EtOH control. F) The relative expression of ESR1 (ERα) in MCF-7 cells treated as in panel D. CT values are shown as avg. ± SEM of 3 replicates in one experiment. The inset shows a western blot of ERα protein. The blot was stripped and reprobed for β-actin. The ratio of ERα/β-actin in MCF-7 was set to 1 and the relative expression of ERα in MCF-7 was 0.4 = 60% lower ERα in LY2 compared to MCF-7 cells.

Figure 5. Q-PCR analysis of the miRNA expression in MCF-7 and LY2 cells

A) Cells were treated with EtOH, 10 nM E₂, or 100 nM 4-OHT for 6 h. Where indicated MCF-7 (B) and LY2 (C) cells were pretreated with 100 nM ICI 182,780 for 6 h. Values are the average of 3–8 separate experiments were normalized by U38 or 5S rRNA and are expressed as fold relative to EtOH-treated MCF-7 expression for each miRNA (arrows). In A: * Significantly different from EtOH in MCF-7. In B and C: * Significantly different from E₂ or 4-OHT in the absence of ICI.

Figure 6. Time course analysis of miRNA expression

MCF-7 cells serum starved for 72 h and were treated for 1, 4, 6 and 8 h with EtOH, 10 nM E₂, or 100 nM 4-OHT. Values are the avg. ± SEM of 3–6 separate experiments in which each point was run in triplicate. Values were normalized by 5S rRNA and are expressed as fold relative to basal (time 0).

Figure 7. Time-dependent changes in ERα, ERβ, and Ago2 expression in E₂- or 4-OHT-treated MCF-7 cells

MCF-7 cells were serum-starved for 48 h and not treated (No Tx) or treated for 1, 4, or 6 h with EtOH, 10 nM E₂, or 10 nM 4-OHT. A) WCE (30 μg protein) were separated on SDS-PAGE gels and western blotted with two different ERα antibodies (D-12 and AER320), ERβ antibody H150, or an antibody for Ago2. The blot was stripped and re-probed for α-tubulin as a loading control. The values below each blot are the ratio of the indicated protein/α-tubulin normalized to the No Tx control. The last lane shows MCF-7 cells that were not serum-starved or treated. B) The relative expression of miR-21 in MCF-7 cells treated as indicated are plotted with ERα (AER320) and ERβ protein expression. The miR-21 data are the same as in Figure 6.

Figure 8. Computational identification of mRNA gene targets of 12 miRNAs oppositely expressed in MCF-7 and LY2 cells

Target prediction software was used to identify mRNA targets of the miRNAs. Predicted genes were overlapped with microarray data of 4-OHT regulated genes by [64]. This identified 36 gene targets as indicated.

Figure 9. miR-21 target genes expression in MCF-7 and LY2 cells

MCF-7 and LY2 cells were serum-starved for 48 h and then treated with EtOH, 10 nM E₂, or 100 nM 4-OHT for 6 h prior to RNA isolation (A) or 24 h prior to WCE preparation (B) as described in Materials and Methods. (A) Q-PCR was performed for the indicated genes and fold-expression determined compared to EtOH as described in Materials and Methods. Values are the average of 3 separate determinations ± SEM. (B) Western blot for the indicated proteins. The membrane was stripped and reprobed for α-tubulin for normalization as described in Materials and Methods. The blot shown is representative of three separate biological replicates.

Figure 10. CYP1B1, ERBB3, and ESR1 gene expression in MCF-7 and LY2 cells

MCF-7 and LY2 cells were serum-starved for 48 h and then treated with EtOH, 10 nM E₂, or 100 nM 4-OHT for 6 h prior to RNA isolation (A) or 24 h prior to WCE preparation (B) as described in Materials and Methods. Q-PCR was performed for the indicated genes and fold-expression determined compared to EtOH as described in Materials and Methods. Values are the average of 3 separate determinations ± SEM.

Figure 11. ZEB1 and E-cadherin expression

Whole cell lysates or nuclear extracts were prepared from the indicated breast cancer cell lines. Identical amounts (30 μg) of protein were immunoblotted for ZEB1 and E-cadherin as described in Materials and Methods. The membranes were stripped and reprobed for β-actin. These blots are representative of three separate experiments.