Zahraei Mahyar, Azimi Yasamin, Karimipour Morteza, Rahimi-Jamnani Fatemeh, Valizadeh Vahideh, Azizi Masoumeh
Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran, Islamic Republic of.
Student Research Committee, Pasteur Institute of Iran, Tehran, Iran.
Sci Rep. 2025 Aug 28;15(1):31739. doi: 10.1038/s41598-025-16911-8.
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.
癌症进展通常伴随着肿瘤抑制性微小RNA（如miR - 200c）的表观遗传沉默，miR - 200c是上皮-间质转化（EMT）和转移的关键调节因子。鉴于DNA甲基化的可逆性，我们采用CRISPR/dCas9 - TET1系统靶向miR - 200c启动子，并在MCF - 7和MDA - MB - 231乳腺癌细胞系中恢复其表达。设计了两个引导RNA（gRNA）位于miR - 200c启动子富含CpG的区域两侧，单独或联合递送它们可实现位点特异性去甲基化。同时转染两个gRNA导致miR - 200c表达协同增加，这可能是由于dCas9 - TET1募集覆盖范围扩大所致。这种上调导致关键的EMT相关转录因子ZEB1、ZEB2以及癌基因KRAS的下调，同时MDA - MB - 231细胞中E - 钙黏蛋白表达增加。然而，MCF - 7细胞中E - 钙黏蛋白的变化很小，这突出了表观遗传调控的复杂性和背景依赖性。功能分析进一步证实了miR - 200c恢复的抗肿瘤作用，细胞活力降低，凋亡增加，在最初表现出较高miR - 200c启动子甲基化的MDA - MB - 231细胞中这些作用更明显。总的来说，我们的研究结果表明，CRISPR/dCas9 - TET1介导的表观遗传编辑有效地重新激活了miR - 200c，逆转了与EMT相关的基因表达，并损害了肿瘤细胞的侵袭性，支持了其作为乳腺癌靶向治疗策略的潜力。
