CRISPR/dCas9-TET1–Mediated Epigenetic Editing Reactivates miR-200c in Breast Cancer Cells

Aberrant DNA methylation of tumor-suppressive microRNAs is a frequent epigenetic alteration driving cancer progression. This study explores the targeted demethylation of the miR-200c promoter, a key regulator of epithelial–mesenchymal transition (EMT), using the CRISPR/dCas9-TET1 system in breast cancer cell lines. Two specific sgRNAs were designed to guide the dCas9-TET1 construct to the miR-200c promoter in MCF-7 and MDA-MB-231 cells. High-Resolution Melting (HRM) analysis showed decreased promoter methylation around 50% in MDA-MB-231 and moderately in MCF-7. Quantitative PCR confirmed a significant increase in miR-200c expression, with combined sgRNAs producing a synergistic effect in MCF-7, while only sgRNA1 was effective in MDA-MB-231. The increased expression of miR-200c led to downregulation of its targets ZEB1, ZEB2, and KRAS. Functionally, demethylation reduced cell proliferation and induced apoptosis, as shown by MTT assays and apoptosis analysis. These findings suggest that CRISPR/dCas9-TET1-mediated epigenetic editing can reactivate silenced miR-200c and inhibit malignant traits in breast cancer cells. This approach highlights a promising therapeutic strategy targeting the epigenome to restore tumor-suppressive functions.