Wang Peiyan, Yu Xiaofei, Sun Pei, Pan Keqing, Sun Jian, Guo Yiqing, Liu Zhaochen, Jiao Mengyu, Deng Jing, Zhang Hui
Department of Stomatology, The Affiliated Hospital of Qingdao University, Qingdao, China.
School of Stomatology, Qingdao University, Qingdao, China.
J Oral Pathol Med. 2025 Jan;54(1):39-48. doi: 10.1111/jop.13593. Epub 2024 Dec 12.
The inactivation of tumor suppressor genes (TSGs) caused by abnormal DNA methylation is confirmed to be widely present in oral potential malignant diseases (OPMDs). Carotenoids like lycopene and astaxanthin can regulate DNA methylation and exert anticancer effects. Therapeutic effect of astaxanthin in OPMDs and oral squamous cell carcinoma (OSCC) models is confirmed, but the relationship between the anti-cancer ability of astaxanthin and its DNA methylation regulation ability remains unclear.
Whole-genome bisulfite sequencing (WGBS) were used to provide biological information associated with DNA methylation. Methylation specific PCR was used to detect the methylation level of specific sites. Related markers were evaluated by qRT-PCR and western blot. CCK8 assay, cell scratch assay, flow cytometric analysis were performed to investigate the cell viability, migration, cell cycle, and apoptosis after treated with concentrations of astaxanthin.
WGBS revealed that HOXA3 and SOX1 were the TSGs with significant differences in promoter CpG methylation of oral dysplastic keratinocytes (DOK) cells. After treatment with 8 μM astaxanthin, the promoter CpG methylation levels of the TSGs were significantly reduced, resulting in the increase in gene expression. The overall effect of astaxanthin on DOK cells is inhibiting cell viability, reducing cell migration, leading to cell cycle G/G arrest, and promoting apoptosis.
This study confirmed significant differences in DNA methylation patterns among oral normal, dysplastic, and cancerous cells. Astaxanthin can reduce the promoter CpG methylation level of TSGs by reducing DNA methyltransferase 1 protein expression level, upregulating mRNA and protein expression, and subsequently modulating the biological behavior of DOK.
由异常DNA甲基化导致的肿瘤抑制基因（TSGs）失活已被证实在口腔潜在恶性疾病（OPMDs）中广泛存在。类胡萝卜素如番茄红素和虾青素可调节DNA甲基化并发挥抗癌作用。虾青素在OPMDs和口腔鳞状细胞癌（OSCC）模型中的治疗效果已得到证实，但其抗癌能力与其DNA甲基化调节能力之间的关系仍不清楚。
采用全基因组亚硫酸氢盐测序（WGBS）提供与DNA甲基化相关的生物学信息。甲基化特异性PCR用于检测特定位点的甲基化水平。通过qRT-PCR和蛋白质免疫印迹法评估相关标志物。进行CCK8检测、细胞划痕试验、流式细胞术分析，以研究不同浓度虾青素处理后细胞的活力、迁移、细胞周期和凋亡情况。
WGBS显示HOXA3和SOX1是口腔发育异常角质形成细胞（DOK）中启动子CpG甲基化存在显著差异的TSGs。用8μM虾青素处理后，TSGs的启动子CpG甲基化水平显著降低，导致基因表达增加。虾青素对DOK细胞的总体作用是抑制细胞活力、减少细胞迁移、导致细胞周期G/G期阻滞并促进凋亡。
本研究证实了口腔正常、发育异常和癌细胞之间DNA甲基化模式存在显著差异。虾青素可通过降低DNA甲基转移酶1蛋白表达水平，上调mRNA和蛋白表达，进而降低TSGs的启动子CpG甲基化水平，随后调节DOK的生物学行为。
