Department of Animal Biology, Faculty of Sciences, Damascus University, Damascus, Syria.
Human Genetics Division, Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), P.O. Box 6091, Damascus, Syria.
Sci Rep. 2022 May 6;12(1):7494. doi: 10.1038/s41598-022-11634-6.
Ovarian cancer is one of the lethal gynecologic cancers. Chemoresistance is an essential reason for treatment failure and high mortality. Emerging evidence connects epithelial-mesenchymal transition (EMT) like changes and acquisition of chemoresistance in cancers. Including EMT, DNA methylation influences cellular processes. Here, EMT-like changes were investigated in cisplatin-resistant A2780 ovarian cancer cells (A2780cis), wherein role of DNA methylation in some EMT genes regulations was studied. Cell viability assay was carried out to test the sensitivity of A2780, and A2780cis human cancer cell lines to cisplatin. Differential mRNA expression of EMT markers using qPCR was conducted to investigate EMT like changes. CpG methylation role in gene expression regulation was investigated by 5-azacytidine (5-aza) treatment. DNA methylation changes in EMT genes were identified using Methylscreen assay between A2780 and A2780cis cells. In order to evaluate if DNA methylation changes are causally underlying EMT, treatment with 5-aza followed by Cisplatin was done on A2780cis cells. Accordingly, morphological changes were studied under the microscope, whereas EMT marker's gene expression changes were investigated using qPCR. In this respect, A2780cis cell line has maintained its cisplatin tolerance ability and exhibits phenotypic changes congruent with EMT. Methylscreen assay and qPCR study have revealed DNA hypermethylation in promoters of epithelial adhesion molecules CDH1 and EPCAM in A2780cis compared to the cisplatin-sensitive parental cells. These changes were concomitant with gene expression down-regulation. DNA hypomethylation associated with transcription up-regulation of the mesenchymal marker TWIST2 was observed in the resistant cells. Azacytidine treatment confirmed DNA methylation role in regulating gene expression of CDH1, EPCAM and TWIST2 genes. A2780cis cell line undergoes EMT like changes, and EMT genes are regulated by DNA methylation. To that end, a better understanding of the molecular alterations that correlate with chemoresistance may lead to therapeutic benefits such as chemosensitivity restoration.
卵巢癌是致命的妇科癌症之一。化疗耐药是治疗失败和高死亡率的一个重要原因。新出现的证据将上皮-间充质转化 (EMT) 样变化和癌症获得化疗耐药联系起来。包括 EMT 在内，DNA 甲基化影响细胞过程。在这里，研究了顺铂耐药 A2780 卵巢癌细胞 (A2780cis) 中的 EMT 样变化，研究了 DNA 甲基化在一些 EMT 基因调控中的作用。通过细胞活力测定法测试 A2780 和 A2780cis 人癌细胞系对顺铂的敏感性。使用 qPCR 进行 EMT 标志物的差异 mRNA 表达研究，以研究 EMT 样变化。通过 5-氮杂胞苷 (5-aza) 处理研究 CpG 甲基化在基因表达调控中的作用。使用 Methylscreen assay 在 A2780 和 A2780cis 细胞之间鉴定 EMT 基因中的 DNA 甲基化变化。为了评估 DNA 甲基化变化是否是 EMT 的因果关系，对 A2780cis 细胞进行了 5-aza 处理后再用顺铂处理。相应地，在显微镜下研究形态变化，同时使用 qPCR 研究 EMT 标志物的基因表达变化。在这方面，A2780cis 细胞系保持了其对顺铂的耐受性能力，并表现出与 EMT 一致的表型变化。Methylscreen assay 和 qPCR 研究表明，与顺铂敏感的亲本细胞相比，A2780cis 中的上皮黏附分子 CDH1 和 EPCAM 的启动子中存在 DNA 超甲基化。这些变化伴随着基因表达下调。在耐药细胞中观察到与间充质标记物 TWIST2 的转录上调相关的 DNA 去甲基化。阿扎胞苷处理证实了 DNA 甲基化在调节 CDH1、EPCAM 和 TWIST2 基因表达中的作用。A2780cis 细胞系发生 EMT 样变化，EMT 基因受 DNA 甲基化调控。为此，更好地了解与化疗耐药相关的分子改变可能会带来治疗益处，例如恢复化疗敏感性。
