Urrutia Guillermo, Laurito Sergio, Campoy Emanuel, Nasif Daniela, Branham María Teresita, Roqué María
IHEM, National University of Cuyo, CONICET, Mendoza, Argentina. Email:
Asian Pac J Cancer Prev. 2018 Oct 26;19(10):2859-2866. doi: 10.22034/APJCP.2018.19.10.2859.
Objective: Breast cancer is a heterogeneous disease characterized by an accumulation of genetic and epigenetic alterations that lead tumor cells to acquire characteristics like the capacity for invasion and metastasis. Metastasis remains a major challenge in cancer management and understanding of its molecular basis should result in improved prevention, diagnosis, and treatment of breast cancer patients. The aim of this study was to investigate how promoter DNA methylation regulates PAX6 gene expression and influences breast carcinoma cell migration. Methods: PAX6 promoter methylation was detected by Methyl Specific-Multiplex Ligation Probe Amplification (MS-MLPA). Gene expression was evaluated using qRT-PCR, while the effect of PAX6 on migration was ssessed by wound healing assay. In addition, MMP2 and MMP9 genes were studied using different bioinformatic tools. Results: The PAX6 promoter is methylated in breast cancer cell lines and methylation in this region impacts on its expression. Migration assays revealed that PAX6 overexpression promotes cell migration, while PAX6 inhibition decreases it. More importantly, we found that migration is affected by PAX6 methylation status. Employing bioinformatic analysis, binding sites for PAX6 on the regulatory regions of the MMP2 and MMP9 genes were established, PAX6 overexpression increasing MMP2 and MMP9 expression at the mRNA level. Conclusion: Our study provides novel insights into epigenetic events that regulate PAX6 expression and molecular mechanisms by which PAX6 modifies the migration capacity of breast cancer cells.
乳腺癌是一种异质性疾病，其特征是遗传和表观遗传改变的积累，这些改变导致肿瘤细胞获得侵袭和转移能力等特征。转移仍然是癌症治疗中的一个主要挑战，对其分子基础的了解应有助于改善乳腺癌患者的预防、诊断和治疗。本研究的目的是探讨启动子DNA甲基化如何调节PAX6基因表达并影响乳腺癌细胞迁移。方法：采用甲基化特异性多重连接探针扩增技术（MS-MLPA）检测PAX6启动子甲基化。使用qRT-PCR评估基因表达，同时通过伤口愈合试验评估PAX6对迁移的影响。此外，使用不同的生物信息学工具研究MMP2和MMP9基因。结果：PAX6启动子在乳腺癌细胞系中发生甲基化，该区域的甲基化影响其表达。迁移试验表明，PAX6过表达促进细胞迁移，而PAX6抑制则降低细胞迁移。更重要的是，我们发现迁移受PAX6甲基化状态的影响。通过生物信息学分析，确定了PAX6在MMP2和MMP9基因调控区域的结合位点，PAX6过表达在mRNA水平上增加MMP2和MMP9的表达。结论：我们的研究为调节PAX6表达的表观遗传事件以及PAX6改变乳腺癌细胞迁移能力的分子机制提供了新的见解。
