Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jan 22:6-7:100024.
doi: 10.1016/j.mbplus.2020.100024. eCollection 2020 May.

miR-200b restrains EMT and aggressiveness and regulates matrix composition depending on ER status and signaling in mammary cancer

Zoi Piperigkou  1   2 , Marco Franchi  3 , Christoph Riethmüller  4 , Martin Götte  2 , Nikos K Karamanos  1   5
Affiliations

miR-200b restrains EMT and aggressiveness and regulates matrix composition depending on ER status and signaling in mammary cancer

Zoi Piperigkou et al. Matrix Biol Plus. .

Abstract

Secreted microRNAs (miRNAs) reside in a complex regulatory network with extracellular matrix (ECM) macromolecules, which affect cell-cell communication, therefore miRNA expression highlights its significance in several aspects of human diseases, including cancer. miRNA-mediated regulation of breast cancer has received considerable attention due to evidence that shows miRNAs to mediate estrogen receptor (ER) status, metastasis, chemoresistance and epithelial-to-mesenchymal transition (EMT). miR-200b is a pluripotent miRNA, which is inversely regulated by ERα and ERβ in mammary cancer. It has been identified as tumor suppressor and EMT inhibitor serving as a critical biomarker, as its expression in breast tumor determines the disease-free survival, thus highlighting its roles in breast cancer invasion and metastasis. The main goal of this study was to investigate the role of miR-200b in modulating the behavior of breast cancer cells with different ER status. We demonstrate that estrogen signaling through ERs reduces miR-200b expression levels in ERα-positive breast cancer cells. Moreover, miR-200b upregulation reduces the aggressive phenotype of ERβ-positive breast cancer cells by inhibiting cell invasiveness and motility, followed by ECM reorganization as well as cytoskeletal and morphological changes concluded from deep inspection of cell topography. Future investigation towards the mechanistic perspective of miR-200b effects in the behavior of aggressive mammary cancer cells appears rewarding in order to expand our understanding of miR-200b as a novel mediator beyond breast cancer diagnosis and pharmaceutical targeting.

Keywords: Breast cancer; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; EMT, epithelial-to-mesenchymal-transition; ER, estrogen receptor; Erk, extracellular signal-regulated kinase; Estrogen receptors; Extracellular matrix; GAG, glycosaminoglycan; GF, growth factor; HER2, human epidermal growth factor receptor 2; IGF-IR, insulin-like growth factor receptor type I; IL, interleukin; MMP, matrix metalloproteinase; PG, proteoglycan; PR, progesterone receptor; RISC, RNA-induced silencing complex; SERM, selective estrogen receptor modulator; TGFβ, transforming growth factor beta; miR-200b; miRNA, microRNA; miRNAs; pre-miRNA, precursor miRNA.

PubMed Disclaimer

Conflict of interest statement

C.R. is an employee of Serend-ip GmbH; the other authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
miR-200b expression is regulated by ER status and signaling and is correlated with better prognosis in breast cancer patients. (A) Tamoxifen treatment results in a downregulation of miR-200b expression in ERα-positive breast cancer cells, MCF-7. MCF-7 cells were treated with 100 nM tamoxifen for 24 h, followed by real-time qPCR analysis of miR-200b expression. (B) Kaplan-Meier survival analysis of human miR-200b (hsa-miR-200b) in breast cancer patients following systemic treatment. P value and hazard ratio (HR) value were calculated using a log-rank test [35]. (C) ΜCF-7 and MDA-MB-231 cells were transfected with a control miRNA (control miR) or a miR-200b precursor (pre-miR-200b) and the overexpression of miR-200b gene was monitored with real-time qPCR analysis. Expression was normalized to 18S rRNA expression. Asterisks (*), (**) indicate statistically significant differences (p ≤ 0.05 and p ≤ 0.01, respectively).
Fig. 2
Fig. 2
Functional analysis of miR-200b upregulation on invasiveness, viability and migration of MCF-7 and MDA-MB-231 breast cancer cells. Cells were transfected with a control miRNA (control miR) or pre-miR-200b. (A) miR-200b overexpression significantly inhibited MDA-MB-231 breast cancer cell invasiveness. Right panel: representative micrographs of invasion filter membranes after crystal violet staining. (B) miR-200b reduces cell growth and (C) migration after 24 h of ΜDA-MB-231 breast cancer cells. Asterisks (*), (**) indicate statistically significant differences (p ≤ 0.05 and p ≤ 0.01, respectively).
Fig. 3
Fig. 3
miR-200b overexpression results in altered morphology and regulates cytoskeletal structures in MDA-MB-231 breast cancer cells. (A) Cell morphology was monitored by SEM analysis. miR-200b overexpression resulted in less cytoplasmic protrusions and more cell-cell contacts (arrow) in ΜDA-MB-231 cells, whereas the morphology of MCF-7 cells was not affected by pre-miR-200b. (B) Immunofluorescence analysis of F-actin (red) in MCF-7 and ΜDA-MB-231 cells with or without pre-miR-200b (scale bar ~10 μm). (C, D) Nano-texture analysis. MDA-MB-231 cell surfaces transfected with control miR (left column) or with pre-miR-200b (right column) were imaged by AFM at nanometer resolution. Shown are the overlay of topography raw data of a 10 μm2 scan and protruding structure elements (green) (C), and (D) the number values for the object depth and the total volume [given as the sum of individual sizes (LDVs, local deviational volumes) per image]. Asterisks (**), (***) indicate statistically significant differences (p ≤ 0.01 and p ≤ 0.0001, respectively).
Fig. 4
Fig. 4
The overexpression of miR-200b inhibits in EMT process in MDA-MB-231 breast cancer cells. (A, B) Immunofluorescence analysis of E-cadherin (green) and vimentin (green) in MCF-7 and ΜDA-MB-231 cells with or without pre-miR-200b (scale bar ~10 μm). (C) Fibronectin, Snail2/Slug and ZEB2 are predicted targets of miR-200b. An alignment of the miR-200b seed sequence with the 3′UTRs of these mRNAs was performed utilizing the public target prediction database microRNA.org. Number indicate the corresponding base pairs in the target mRNA sequence. (D) mRNA Expression of the epithelial marker E-cadherin and the mesenchymal markers fibronectin, Snail2/Slug and ZEB2 in MCF-7 and ΜDA-MB-231 cells with or without pre-miR-200b, as determined by qPCR. Asterisks (*), (**) indicate statistically significant differences (p ≤ 0.05 and p ≤ 0.01, respectively).
Fig. 5
Fig. 5
miR-200b regulates matrix composition and signaling in MDA-MB-231 breast cancer cells. Real-time qPCR analysis of major MMPs (MMP2, MMP7, MMP9, MT1-MMP) (A), (B) MMP2/MMP9 gelatinolytic activities (as assayed by gelatin zymography), and expression (mRNA and protein) of the cell membrane PG, syndecan-1 (C, D), following miR-200b overexpression in MCF-7 and ΜDA-MB-231 cells with real-time qPCR analysis and immunofluorescence microscopy. The mRNA levels were evaluated using β-actin as reference gene. (E) Immunoblots of phospho-Erk1/2, total-Erk1/2 and α-tubulin in MCF-7 and ΜDA-MB-231 cells in the presence or absence of pre-miR-200b. Asterisks (*), (**) indicate statistically significant differences (p ≤ 0.05 and p ≤ 0.01, respectively).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Torre L.A., Bray F., Siegel R.L., Ferlay J., Lortet-Tieulent J., Jemal A. Global cancer statistics, 2012. CA Cancer J. Clin. 2015;65:87–108. - PubMed
    1. Dai X., Xiang L., Li T., Bai Z. Cancer hallmarks, biomarkers and breast cancer molecular subtypes. J. Cancer. 2016;7:1281–1294. - PMC - PubMed
    1. Gong Y., Liu Y.R., Ji P., Hu X., Shao Z.M. Impact of molecular subtypes on metastatic breast cancer patients: a SEER population-based study. Sci. Rep. 2017;7 - PMC - PubMed
    1. Espinoza-Sanchez N.A., Gotte M. Role of cell surface proteoglycans in cancer immunotherapy. Semin Cancer Biol. 2019 doi: 10.1016/j.semcancer.2019.07.012. - DOI - PubMed
    1. Liang Y., Zhang H., Song X., Yang Q. Metastatic heterogeneity of breast cancer: molecular mechanism and potential therapeutic targets. Semin Cancer Biol. 2019 doi: 10.1016/j.semcancer.2019.08.012. - DOI - PubMed