Open Access

Functional impact of the long non‑coding RNA MEG3 deletion by CRISPR/Cas9 in the human triple negative metastatic Hs578T cancer cell line

  • Authors:
    • Carlos Deocesano‑Pereira
    • Raquel Arminda Carvalho Machado
    • Henrique Cesar de Jesus‑Ferreira
    • Thiago Marchini
    • Tulio Felipe Pereira
    • Ana Claudia Oliveira Carreira
    • Mari Cleide Sogayar
  • View Affiliations

  • Published online on: October 8, 2019     https://doi.org/10.3892/ol.2019.10969
  • Pages: 5941-5951
  • Copyright: © Deocesano‑Pereira et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Long non‑coding RNAs (lncRNAs) serve critical roles in regulating cellular homeostasis, and their deregulated expression/activity is associated with neoplastic transformation. The maternally expressed gene 3 (MEG3) has been extensively described as a tumor suppressor gene in different types of cancer, including breast cancer. Interestingly, using a panel of seven different breast cancer cell lines, the present study revealed that MEG3 is highly expressed in the triple negative metastatic human Hs578T breast cancer cell line, which is refractory to different therapeutic approaches. Therefore, the present study aimed to investigate the phenotypic impact of MEG3 deletion in this cell line. Using the CRISPR/Cas9 system, complete knockout (KO) of MEG3 was achieved. Deletion was confirmed by genomic PCR and reverse transcription‑quantitative PCR. The MEG3_KO cell population displaying the highest efficiency of genomic editing was selected for phenotypic in vitro assays, including wound scratch and Transwell assays, flow cytometry and immunofluorescence. The results demonstrated that MEG3 deletion increased cell proliferation, anchorage‑independent cell growth and cell motility, which was consistent with its well‑known tumor suppressor function. However, the present study revealed that MEG3_KO also lead to decreased cell invasiveness ability, supporting previous evidence that MEG3 modulates epithelial‑to‑mesenchymal inducing factors. The present study demonstrated that deletion of MEG3 promoted an increase in transforming growth factor β and N‑cadherin protein levels and significant reduction in matrix metallopeptidase 2, zinc‑finger E‑box binding homeobox 1 and collagen type III α1 chain gene expression levels. Additionally, MEG3_KO cells displayed significant resistance to doxorubicin treatment, demonstrating the role of this lncRNA in cancer cell survival by regulating apoptosis. The present study highlighted the utility of CRISPR/Cas9 for anticancer studies of intergenic lncRNAs and demonstrated that, although Hs578T cells express MEG3 at high levels, these cells display mechanisms to escape the growth suppression effects of this lncRNA. Notably, the detailed pathological mechanisms of MEG3 concerning tumor metastasis remain to be elucidated prior to applying MEG3 expression/activation in future therapeutic approaches for breast cancer treatment.

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December 2019
Volume 18 Issue 6

Print ISSN: 1792-1074
Online ISSN:1792-1082

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Copy and paste a formatted citation
APA
Deocesano‑Pereira, C., Machado, R.A., de Jesus‑Ferreira, H.C., Marchini, T., Pereira, T.F., Carreira, A.C., & Sogayar, M.C. (2019). Functional impact of the long non‑coding RNA MEG3 deletion by CRISPR/Cas9 in the human triple negative metastatic Hs578T cancer cell line. Oncology Letters, 18, 5941-5951. https://doi.org/10.3892/ol.2019.10969
MLA
Deocesano‑Pereira, C., Machado, R. A., de Jesus‑Ferreira, H. C., Marchini, T., Pereira, T. F., Carreira, A. C., Sogayar, M. C."Functional impact of the long non‑coding RNA MEG3 deletion by CRISPR/Cas9 in the human triple negative metastatic Hs578T cancer cell line". Oncology Letters 18.6 (2019): 5941-5951.
Chicago
Deocesano‑Pereira, C., Machado, R. A., de Jesus‑Ferreira, H. C., Marchini, T., Pereira, T. F., Carreira, A. C., Sogayar, M. C."Functional impact of the long non‑coding RNA MEG3 deletion by CRISPR/Cas9 in the human triple negative metastatic Hs578T cancer cell line". Oncology Letters 18, no. 6 (2019): 5941-5951. https://doi.org/10.3892/ol.2019.10969