Laboratory of Epigenetics, Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
Department of Pathology, Seoul National University College of Medicine, 103 Daehak-ro, Ihwa-dong, Jongno-gu, Seoul, 03080, South Korea.
Gastric Cancer. 2021 May;24(3):602-610. doi: 10.1007/s10120-020-01146-5. Epub 2021 Jan 2.
Gastric cancer (GC) is a leading cause of cancer morbidity and mortality worldwide. This is due to the heterogeneous features of GC, which consist of a diverse molecular phenotype. Epstein-Barr virus (EBV)-positive GC and microsatellite instability (MSI)-high GC encompass similar epigenetic traits, including high levels of DNA methylation in CpG islands; however, EBV-positive and MSI-high GCs are mutually exclusive. We aimed to elucidate the underlying mechanism of this exclusivity.
We knocked out MLH1 in EBV-positive GC cell lines SNU-719 and NCC24 via CRISPR-Cas9, and evaluated the modified cellular properties in vitro and in vivo. The MSI status of each cell line was screened with two marker capillary electrophoresis, and further diagnosed with five marker capillary electrophoresis and parallel sequencing using 21 markers.
Initial evaluation showed that cell growth, migration, invasion, and MSI status were not affected by MLH1 silencing. However, with prolonged passage, GC cell lines gradually gained MSI and NCC24 cells were transformed to EBV-positive/MSI-high GC cells after 12 months. Furthermore, MLH1 silencing reduced tumor stemness in SNU-719 and NCC24 regardless of the MSI status in vitro and in vivo.
Our findings suggest that EBV-positivity and MSI-high status are mutually exclusive due to the immediate disadvantage in tumor stemness when MLH1 is silenced, whereas the establishment of MSI-high status in EBV-positive GCs required a longer period.
胃癌（GC）是全球癌症发病率和死亡率的主要原因。这是由于 GC 的异质性特征，其包含不同的分子表型。EB 病毒（EBV）阳性 GC 和微卫星不稳定（MSI）高 GC 包含相似的表观遗传特征，包括 CpG 岛中高水平的 DNA 甲基化；然而，EBV 阳性和 MSI 高 GC 是相互排斥的。我们旨在阐明这种排他性的潜在机制。
我们通过 CRISPR-Cas9 在 EBV 阳性 GC 细胞系 SNU-719 和 NCC24 中敲除 MLH1，并评估体外和体内修饰后的细胞特性。用两种标记物毛细管电泳筛选每个细胞系的 MSI 状态，并进一步用 21 个标记物的五标记物毛细管电泳和并行测序进行诊断。
初步评估表明，细胞生长、迁移、侵袭和 MSI 状态不受 MLH1 沉默的影响。然而，随着传代时间的延长，GC 细胞系逐渐获得 MSI，NCC24 细胞在 12 个月后转化为 EBV 阳性/ MSI 高 GC 细胞。此外，MLH1 沉默降低了 SNU-719 和 NCC24 中的肿瘤干性，无论其体外和体内的 MSI 状态如何。
我们的研究结果表明，由于 MLH1 沉默时肿瘤干性的即时劣势，EBV 阳性和 MSI 高状态是相互排斥的，而 EBV 阳性 GCs 中 MSI 高状态的建立需要更长的时间。
