Department of Respiratory Medicine, Yue Bei People's Hospital, Shaoguan, China.
Central Laboratory, Yue Bei People's Hospital, Shaoguan, China.
Cell Biol Int. 2022 Nov;46(11):1900-1914. doi: 10.1002/cbin.11868. Epub 2022 Aug 21.
Ras-association domain family 1A (RASSF1A) is one of the most methylated genes in lung cancer (LC). We investigate whether the high DNA methylation level of RASSF1A can relieve the resistance of RASSF1A to LC by inhibiting RASSF1A's transcription factor binding to RASSF1A. RASSF1A expression in tissues and cells was tested utilizing quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. RASSF1A expression and RASSF1A methylation level in LC cells exposed to 5-Aza-dc were assessed by qRT-PCR and quantitative methylation-specific PCR. The association between CTCF and RASSF1A was assessed using hTFtarget, ChIP, and luciferase reporter gene analysis. The effects of 5-Aza-dc, CTCF, and RASSF1A on cell biological behaviors and epithelial-mesenchymal transition (EMT)-related markers were assessed by cell function experiments and Western blot. Moreover, we constructed the xenograft tumor and pulmonary nodule metastasis models, and assessed tumor volume and weight. RASSF1A expression and pulmonary nodule metastasis were tested utilizing qRT-PCR, Western blot, and H&E staining. RASSF1A was under-expressed in LC tissues and cells. 5-Aza-dc enhanced RASSF1A level and weakened RASSF1A methylation level in LC cells. RASSF1A silencing neutralized 5-Aza-dc-mediated repressing effects on LC cell biological function and EMT. The loss of CTCF binding to RASSF1A in LC cells was associated with DNA methylation. The effect of 5-Aza-dc on RASSF1A level, LC cell malignant behaviors, and EMT-related factors were strengthened by CTCF upregulation. RASSF1A overexpression suppressed LC tumor growth and pulmonary nodule metastasis in vivo. DNA methylation blocked the modulation of RASSF1A expression by CTCF and relieved the resistance of RASSF1A to LC.
Ras 相关结构域家族 1A（RASSF1A）是肺癌（LC）中甲基化程度最高的基因之一。我们研究了 RASSF1A 的高 DNA 甲基化水平是否可以通过抑制 RASSF1A 的转录因子与 RASSF1A 的结合来缓解 RASSF1A 对 LC 的耐药性。利用实时定量聚合酶链反应（qRT-PCR）和 Western blot 检测组织和细胞中的 RASSF1A 表达。利用 qRT-PCR 和定量甲基化特异性 PCR 检测暴露于 5-Aza-dc 的 LC 细胞中的 RASSF1A 表达和 RASSF1A 甲基化水平。利用 hTFtarget、ChIP 和荧光素酶报告基因分析评估 CTCF 与 RASSF1A 的关联。通过细胞功能实验和 Western blot 评估 5-Aza-dc、CTCF 和 RASSF1A 对细胞生物学行为和上皮间质转化（EMT）相关标志物的影响。此外，我们构建了异种移植肿瘤和肺结节转移模型，并评估了肿瘤体积和重量。利用 qRT-PCR、Western blot 和 H&E 染色检测 RASSF1A 表达和肺结节转移。RASSF1A 在 LC 组织和细胞中低表达。5-Aza-dc 增强了 LC 细胞中的 RASSF1A 水平并减弱了 RASSF1A 甲基化水平。RASSF1A 沉默中和了 5-Aza-dc 对 LC 细胞生物学功能和 EMT 的抑制作用。LC 细胞中 CTCF 与 RASSF1A 的结合缺失与 DNA 甲基化有关。上调 CTCF 增强了 5-Aza-dc 对 RASSF1A 水平、LC 细胞恶性行为和 EMT 相关因子的作用。RASSF1A 过表达抑制了体内 LC 肿瘤的生长和肺结节转移。DNA 甲基化阻断了 CTCF 对 RASSF1A 表达的调节，缓解了 RASSF1A 对 LC 的耐药性。
