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Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Published on: February 21, 2014
MicroRNA expression profiling of endocrine sensitive and resistant breast cancer cell lines
Maitham A Khajah1, Alyaa Al-Ateyah1, Yunus A Luqmani1
1Faculty of Pharmacy, Kuwait University, Safat, 13110, Kuwait.
Background:
MicroRNAs (miRs) regulate gene expression through translation inhibition of target mRNAs. One of the most promising approaches for cancer therapy is through mimicking or antagonizing the action of miRs. In this report, we analyzed the miRnome profile of several human breast cancer cell lines to determine the influence of estrogen receptor (ER) silencing previously shown to result in epithelial to mesenchymal transition (EMT) and enhanced tumor invasion.
Methods:
MicroRNA extracted from MDA-MB-231 (de novo ER-) and ER-silenced (acquired ER-) pII and IM-26 or ER-expressing (YS1.2) siRNA transfected derivatives of MCF7 cells was deep sequenced on Illumina NextSeq500. Respective miRnomes were compared with edgeR package in R and Venny2.1 and target prediction performed with miRTarBase. Mimics and inhibitors of selected differentially expressed miRs associated with EMT mediators (miR-200c-3p targeting ZEB1, miR-449a targeting δ-catenin and miR-29a-3p) were transfected into pII cells and mRNA targets, as well as E-cadherin and keratin 19 (epithelial and mesenchymal markers respectively) were measured using taqman PCR.
Results:
Each cell line expressed about 20% of the total known human miRnome; There was a high degree of similarity between the 3 tested ER-lines. Out of these expressed miRs, 50-60% were significantly differentially expressed between ER- and ER + lines. Transfection of miR-200c-3p mimic into pII cells down regulated ZEB1 and vimentin, and increased E-cadherin and keratin 19 with accompanying morphological changes, and reduced cell motility, reflecting a reversal back into an epithelial phenotype. On the other hand, transfecting pII with miR-449a inhibitor reduced cell invasion but did not induce EMT. Transfecting pII cell line with the mimic or inhibitor of miR-29a-3p showed no change in EMT markers or cell invasion suggesting that the EMT induced by loss of ER function can be reversed by blocking some but not just any random EMT-associated genes.
Conclusions:
These data suggest that differences in miR expression can be exploited not only as mediators (using mimics) and targets (using miR antagonists) for general cancer therapies aimed at regulating either individual or multiple mRNAs, but also to re-sensitize endocrine resistant breast cancers by turning them back into a type that will be susceptible to endocrine agents.
Insights
Estrogen receptor (ER) silencing in breast cancer cells alters microRNA (miR) expression, driving epithelial to mesenchymal transition (EMT). Restoring miR-200c-3p reverses EMT, suggesting miR-based therapies for endocrine-resistant breast cancers.
Area of Science:
- Genomics and Molecular Biology
- Cancer Research
- Epigenetics
Background:
- MicroRNAs (miRs) are key regulators of gene expression, offering therapeutic potential in cancer by mimicking or antagonizing their actions.
- Estrogen receptor (ER) signaling is crucial in breast cancer; ER silencing induces epithelial to mesenchymal transition (EMT), promoting tumor invasion.
- Understanding miRnome profiles in ER-negative versus ER-positive breast cancer cells is vital for developing targeted therapies.
Purpose of the Study:
- To analyze the miRnome profile of human breast cancer cell lines with varying ER status.
- To investigate the influence of ER silencing on miR expression and its association with EMT.
- To evaluate the therapeutic potential of modulating specific miRs to reverse EMT and enhance endocrine sensitivity.
Main Methods:
- Deep sequencing of microRNA (miRnome) from ER-positive (MCF7 derivatives) and ER-negative/silenced (MDA-MB-231, MCF7 derivatives) breast cancer cell lines.
- Bioinformatic analysis using edgeR for differential miR expression and miRTarBase for target prediction.
- Functional validation via transfection of miR mimics/inhibitors (miR-200c-3p, miR-449a, miR-29a-3p) and assessment of EMT markers (E-cadherin, keratin 19, vimentin) and cell invasion.
Main Results:
- Significant differential expression of 50-60% of expressed miRs between ER-positive and ER-negative/silenced breast cancer cell lines.
- Transfection of miR-200c-3p mimic into ER-negative cells reversed EMT, restoring epithelial phenotype (increased E-cadherin, keratin 19; decreased vimentin) and reducing motility.
- Modulation of miR-449a and miR-29a-3p showed partial or no effect on EMT markers and invasion, indicating specific miR roles in reversing ER-loss-induced EMT.
Conclusions:
- Differential miR expression in breast cancer can be therapeutically exploited using mimics or antagonists to regulate gene expression and target mRNAs.
- Targeted miR modulation, specifically miR-200c-3p, can reverse EMT and potentially restore sensitivity to endocrine therapy in resistant breast cancers.
- These findings highlight the potential of miR-based strategies for overcoming endocrine resistance and improving breast cancer treatment outcomes.
