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Impact of miRNAs expression modulation on the methylation status of breast cancer stem cell-related genes

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Abstract

Purpose

Altered miRNAs play a crucial role in the emergence of the breast cancer stem cell (BCSC) phenotype. The interplay between miRNAs and methylation enzymes has been documented. One of the most aggressive breast cancer cell lines, MDA-MB-231, has expressed much more DNMT3B than DNMT3A. This study aims to evaluate the ability of miR-203 restoration and miR-150 inhibition to regulate DNMT3B and DNMT3A to modify the methylation level of BCSC-associated genes.

Methods

MDA-MB-231 cells were transfected with miR-203 mimic or miR-150 inhibitor or DNMT3B siRNA, and downstream analysis was performed by flow cytometry, real-time PCR and Western blotting.

Results

DNMT3A and DNMT3B are regulated both by miR-203a-3p and miR-150-5p. Transfection with miR-203 mimic and miR-150 inhibitor significantly reduced the CD44+CD24− subpopulation and down-regulated the expression of CD44 mRNA by increasing promoter methylation levels. SiRNA knockdown of DNMT3B increased the CD44+CD24− subpopulation and the expression of CD44 and ALDH1A3 by decreasing methylation density. The inhibition of miR-150 down-regulated OCT3/4 and SOX2 expression without affecting methylation levels, while miR-203 restoration and miR-150 inhibition down-regulated NANOG expression by elevating the methylation level. A positive-feedback loop was found between miR-203 and its target DNMT3B, as restoring miR-203 suppressed DNMT3B, while knocking down DNMT3B up-regulated miR-203. The restoration of miR-203 and knockdown of DNMT3B decreased methylation levels and increased the expression of miR-141 and miR-200c.

Conclusions

The study concluded that miR-203 and miR-150 play a role in the regulation of genes involved in BCSC methylation, including other miRNAs, by targeting DNMT3B and DNMT3A.

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Availability of data and material

No studies have been adopted on humans or animals.

Code availability

All the software used were freely available.

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Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author information

Author notes

  1. H. H. El-Osaily

    Present address: Biochemistry Department, Faculty of Pharmacy, Ahram Canadian University, 4th industrial region, 6th of October City, 12585, Giza, Egypt

Authors and Affiliations

  1. Biochemistry Department, Faculty of Pharmacy for Girls, Al-Azhar University, Cairo, 11651, Egypt

    I. H. Ibrahim

  2. Medical Molecular Genetics Department, National Research Centre, Giza, 12622, Egypt

    M. L. Essawi

  3. Molecular Genetics and Enzymology Department, National Research Centre, Giza, 12622, Egypt

    S. M. Salem

Authors
  1. H. H. El-Osaily
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  2. I. H. Ibrahim
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  3. M. L. Essawi
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  4. S. M. Salem
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Contributions

HHE and SMS conducted the experiment, interpreted the results, analyzed the data, and wrote the manuscript. IHI participated in the design of the experiment. MLE revised and edited the manuscript.

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Correspondence to H. H. El-Osaily.

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El-Osaily, H.H., Ibrahim, I.H., Essawi, M.L. et al. Impact of miRNAs expression modulation on the methylation status of breast cancer stem cell-related genes. Clin Transl Oncol 23, 1440–1451 (2021). https://doi.org/10.1007/s12094-020-02542-0

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  • DOI: https://doi.org/10.1007/s12094-020-02542-0

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