The Central Laboratory of Cancer Hospital of Shantou University Medical College, 7 Raoping Road, Shantou, 515031, China; Present Address: Health Science Center of Shenzhen University Medical College, Shenzhen, 518055, China.
The Central Laboratory of Cancer Hospital of Shantou University Medical College, 7 Raoping Road, Shantou, 515031, China.
Exp Cell Res. 2020 Jan 15;386(2):111743. doi: 10.1016/j.yexcr.2019.111743. Epub 2019 Nov 23.
Notch3 can act as a tumor suppressor in the breast cancer epithelial cells. Unfortunately, Notch3 expression is decreased or lost, especially in triple-negative breast cancer (TNBC) cells, and the reasons remain unclear. Here, we found Notch3 was upregulated in MDA-MB-231 cells with 5-Aza treatment. Two CpG islands were observed in notch3 promoter. Interestingly, bisulfite sequencing exhibited that large amounts of unconverted cytosines were not only followed by guanine, but also adenine, cytosine and thymine, which implied that there simultaneously existed CpG and non-CpG methylation in notch3 promoter. To better analyze the methylation frequency of non-CpG locus, we designed CpG/non-CpG methylation analysis software. The results showed that the methylation frequency of notch3 gene in different breast cancer cell lines was in order T47D, MCF-7, SKBR3, BT-549 and MDA-MB-231. Furthermore, we identified that DNMT3b, DNMT1, DNMT3L, Mecp2 and EZH2 were important regulators of non-CpG locus of notch3 gene. Immunohistochemistry staining revealed a negative correlation between EZH2 and Notch3 from 22 luminal and 26 TNBC cases. In vitro methylation combined luciferase activity assays showed that non-CpG methylation was still crucial cause leading to notch3 transcriptional repression in TNBC. Our findings provide possible explanation for the downregulation or loss of Notch3 expression in TNBC.
Notch3 可作为乳腺癌上皮细胞中的肿瘤抑制因子。不幸的是，Notch3 的表达减少或丢失，尤其是在三阴性乳腺癌（TNBC）细胞中，其原因尚不清楚。在这里，我们发现 Notch3 在经 5-Aza 处理的 MDA-MB-231 细胞中上调。Notch3 启动子中观察到两个 CpG 岛。有趣的是，亚硫酸氢盐测序显示大量未转化的胞嘧啶不仅紧随鸟嘌呤，而且紧随腺嘌呤、胞嘧啶和胸腺嘧啶，这表明 Notch3 启动子中同时存在 CpG 和非 CpG 甲基化。为了更好地分析非 CpG 位点的甲基化频率，我们设计了 CpG/非 CpG 甲基化分析软件。结果表明，不同乳腺癌细胞系中 Notch3 基因的甲基化频率依次为 T47D、MCF-7、SKBR3、BT-549 和 MDA-MB-231。此外，我们确定 DNMT3b、DNMT1、DNMT3L、Mecp2 和 EZH2 是 Notch3 基因非 CpG 位点的重要调节因子。免疫组织化学染色显示 22 例管腔和 26 例 TNBC 病例中 EZH2 与 Notch3 之间呈负相关。体外甲基化结合荧光素酶活性测定表明，非 CpG 甲基化仍然是导致 TNBC 中 Notch3 转录抑制的关键原因。我们的研究结果为 TNBC 中 Notch3 表达下调或缺失提供了可能的解释。
