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Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells
Published on: October 27, 2020
Targeting specificity protein 1 with miR-128-3p overcomes TGF-β1 mediated epithelial-mesenchymal transition in breast
Lakshmi Vineela Nalla1, Piyush Gondaliya2, Kiran Kalia2
1Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research-Ahmedabad (NIPER-A), Palaj, Gandhinagar, Gujarat, 382355, India.
Background:
Specificity protein 1 (SP1) was found to play a critical role in the regulation of TGF-β1 driven epithelial-mesenchymal transition (EMT). Recent clinical findings demonstrated a significant drop in the expression of miR-128-3p with the cancer progression in breast cancer patients. However, the impact of miR-128-3p on the SP1 expression in breast cancer remains unknown. Herein, we evaluated the role of miR-128-3p mimics in suppressing EMT of breast cancer cell lines by regulating the TGF-β1/SP1 axis.
Methods:
miR-128-3p interaction with SP1 was detected by in silico tools and dual-luciferase reporter assay. qPCR, western blot, and immunocytochemistry experiments were conducted for determining the expression levels of miR-128-3p and EMT markers with and without the treatment of miR-128-3p mimics. Further, to understand the effect of miR-128-3p mimics on cancer progression, experiments such as wound healing assay, transwell assay, adhesion assay, and cell cycle analysis were performed.
Results:
A significant inverse relation between SP1 and miR-128-3p levels was found in MCF-7 and MDA-MB-231 cell lines. miR-128-3p overexpression impeded the SP1 mediated EMT markers in TGF-β1 stimulated cells by inhibiting the SP1 nuclear function. Further, treatment with miR-128-3p mimics significantly reduced the migration, invasion and spreading capability of TGF-β1 stimulated cells. Flow cytometry results showed the impeding role of miR-128-3p on the cell cycle progression.
Conclusions:
Upregulated miR-128-3p inhibited SP1, thereby limiting the TGF-β1 induced EMT in MCF-7 and MDA-MB-231 cell lines for the first time. This study may pave the path to explore novel miRNA therapeutics for eradicating advanced breast cancer cases.
Insights
Upregulated miR-128-3p inhibits SP1, suppressing TGF-β1 induced epithelial-mesenchymal transition (EMT) in breast cancer cells. This finding offers potential for novel miRNA therapeutics against advanced breast cancer.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Specificity protein 1 (SP1) is crucial in regulating TGF-β1-induced epithelial-mesenchymal transition (EMT).
- Decreased miR-128-3p expression correlates with breast cancer progression.
- The impact of miR-128-3p on SP1 in breast cancer was previously unknown.
Purpose of the Study:
- To investigate the role of miR-128-3p mimics in suppressing breast cancer cell EMT.
- To determine if miR-128-3p regulates the TGF-β1/SP1 axis in breast cancer.
Main Methods:
- In silico analysis and dual-luciferase reporter assays to confirm miR-128-3p and SP1 interaction.
- qPCR, western blot, and immunocytochemistry to assess expression levels of miR-128-3p and EMT markers.
- Functional assays including wound healing, transwell, adhesion, and cell cycle analysis to evaluate miR-128-3p mimic effects on cancer progression.
Main Results:
- A significant inverse correlation between SP1 and miR-128-3p levels was observed in MCF-7 and MDA-MB-231 cell lines.
- miR-128-3p overexpression inhibited SP1-mediated EMT markers and SP1 nuclear function in TGF-β1-stimulated cells.
- miR-128-3p mimics reduced migration, invasion, and spreading, and impeded cell cycle progression in TGF-β1-stimulated breast cancer cells.
Conclusions:
- Upregulated miR-128-3p effectively inhibits SP1, thereby limiting TGF-β1-induced EMT in breast cancer cell lines.
- This research establishes a novel mechanism for controlling breast cancer progression via the miR-128-3p/SP1 axis.
- The findings suggest potential for developing novel microRNA-based therapeutics for advanced breast cancer treatment.
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