Scholarlink

<p>Cancer progression is often accompanied by epigenetic silencing of tumor-suppressor microRNAs such as<i>miR-200c</i>, a key regulator of epithelial-to-mesenchymal transition (EMT) and metastasis. Given the reversible nature of DNA methylation, we employed a CRISPR/dCas9-TET1 system to target the <i>miR-200c</i> promoter and restore its expression in MCF-7 and MDA-MB-231 breast cancer cell lines. Two gRNAs were designed to flank CpG-rich regions of the <i>miR-200c</i> promoter, and their individual or combined delivery enabled site-specific demethylation. Co-transfection with both gRNAs resulted in a synergistic increase in <i>miR-200c</i> expression, likely due to expanded coverage of dCas9-TET1 recruitment. This upregulation led to the downregulation of key EMT-related transcription factors&#xa0;<i>ZEB1</i>, <i>ZEB2</i>, and the oncogene <i>KRAS</i>, as well as increased <i>E-cadherin</i> expression in MDA-MB-231 cells. However, E-cadherin changes in MCF-7 cells were minimal, highlighting the complex and context-dependent nature of epigenetic regulation. Functional assays further confirmed the anti-tumorigenic effects of&#xa0;<i>miR-200c</i> restoration, with reduced cell viability and increased apoptosis, effects more pronounced in MDA-MB-231 cells, which initially exhibited higher <i>miR-200c</i> promoter methylation. Collectively, our findings demonstrate that CRISPR/dCas9-TET1-mediated epigenetic editing effectively reactivates <i>miR-200c</i>, reverses EMT-associated gene expression, and impairs tumor cell aggressiveness, supporting its potential as a targeted therapeutic strategy in breast cancer.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

CRISPR/dCas9-TET1–mediated epigenetic editing reactivates miR-200c in breast cancer cells

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年8月28日

查看全文
  • Mahyar Zahraei,
  • Yasamin Azimi,
  • Morteza Karimipour,
  • Fatemeh Rahimi-Jamnani,
  • Vahideh Valizadeh,
  • Masoumeh Azizi

摘要

Cancer progression is often accompanied by epigenetic silencing of tumor-suppressor microRNAs such asmiR-200c, a key regulator of epithelial-to-mesenchymal transition (EMT) and metastasis. Given the reversible nature of DNA methylation, we employed a CRISPR/dCas9-TET1 system to target the miR-200c promoter and restore its expression in MCF-7 and MDA-MB-231 breast cancer cell lines. Two gRNAs were designed to flank CpG-rich regions of the miR-200c promoter, and their individual or combined delivery enabled site-specific demethylation. Co-transfection with both gRNAs resulted in a synergistic increase in miR-200c expression, likely due to expanded coverage of dCas9-TET1 recruitment. This upregulation led to the downregulation of key EMT-related transcription factors ZEB1, ZEB2, and the oncogene KRAS, as well as increased E-cadherin expression in MDA-MB-231 cells. However, E-cadherin changes in MCF-7 cells were minimal, highlighting the complex and context-dependent nature of epigenetic regulation. Functional assays further confirmed the anti-tumorigenic effects of miR-200c restoration, with reduced cell viability and increased apoptosis, effects more pronounced in MDA-MB-231 cells, which initially exhibited higher miR-200c promoter methylation. Collectively, our findings demonstrate that CRISPR/dCas9-TET1-mediated epigenetic editing effectively reactivates miR-200c, reverses EMT-associated gene expression, and impairs tumor cell aggressiveness, supporting its potential as a targeted therapeutic strategy in breast cancer.