■经典抑癌基因p16的异常表达是肺癌中的常见事件,主要是由于其5'-胞嘧啶-磷酸-鸟嘌呤-3'岛(Cgi)的高度甲基化。然而,甲基化是否发生在其他区域以及p16表达和功能如何受到影响在很大程度上是未知的。
■聚簇定期间隔短回文重复/dCas9(CRISPR/dCas9)技术用于p16特定位点的甲基化编辑。3-(4,5-二甲基噻唑-2-基)-5(3-羧基甲氧基苯基)-2-(4-磺基苯基)-2H-四唑鎓检测到甲基化编辑的影响,内盐(MTS),Transwell迁移和伤口愈合试验。进行染色质免疫沉淀定量聚合酶链反应(CHIP-qPCR)以探讨Cgi岸上甲基化对转录因子(TFs)包括YY1,SP1,ZNF148和OTX2与p16基因结合能力的影响。进行拯救实验以验证OTX2对p16的调节作用。通过癌症基因组图谱(TCGA)程序和肺腺癌(LUAD)患者样本的数据集,进一步验证了p16表达与非启动子区域Cgishore甲基化水平之间的负相关。
■使用CRISPR/dCas9介导的特异性位点甲基化编辑在HEK293和A549细胞中证明了p16Cgi岸上甲基化对其表达的抑制作用。在p16非启动子区域的Cgi岸上的甲基化显着降低了其表达并促进了细胞生长和迁移。甲基化修饰后,OTX2与p16结合的能力显著降低19.35%。OTX2在A549细胞中的过表达部分逆转了甲基化对p16表达的抑制作用19.04%。TCGA和LUAD患者样品的验证结果支持p16Cgishore是一个关键的甲基化调节区。
■我们的研究结果表明,p16非启动子区域的Cgi岸上甲基化可以阻碍OTX2的转录活性,导致p16表达减少,这可能有助于肺癌的发展。
UNASSIGNED: The aberrant expression of the classical tumor suppressor gene p16 is a frequent event in lung cancer mainly due to the hypermethylation of its 5\'-cytosine-phosphate-guanine-3\' island (Cgi). However, whether methylation happens in other regions and how p16 expression and function are affected are largely unknown.
UNASSIGNED: Clustered Regularly Interspaced Short Palindromic Repeats/dCas9 (CRISPR/dCas9) technology was used for methylation editing at specific site of p16. The effects of methylation editing were detected by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt (MTS), transwell migration and wound healing tests. Chromatin immnoprecipitation-quantitative polymerase chain reaction (CHIP-qPCR) was performed to explore the impact of Cgi shore methylation on the binding abilities of transcription factors (TFs) including YY1, SP1, ZNF148 and
OTX2 to p16 gene. A rescue experiment was performed to verify the regulatory effect of
OTX2 on p16. The negative relationship between p16 expression and the methylation level of Cgi shore in non-promoter region was further verified with datasets from The Cancer Genome Atlas (TCGA) program and lung adenocarcinoma (LUAD) patients\' samples.
UNASSIGNED: The suppressive effect of p16 Cgi shore methylation on its expression was demonstrated in both HEK293 and A549 cells using CRISPR/dCas9-mediated specific site methylation editing. Methylation of the Cgi shore in the p16 non-promoter region significantly decreased its expression and promoted cell growth and migration. The ability of
OTX2 bound to p16 was significantly reduced by 19.35% after methylation modification. Over-expression of
OTX2 in A549 cells partly reversed the inhibitory effect of methylation on p16 expression by 19.04%. The verification results with TCGA and LUAD patients\' samples supported that the p16 Cgi shore is a key methylation regulatory region.
UNASSIGNED: Our findings suggested that methylation of the Cgi shore in the p16 non-promoter region can hamper the transcriptional activity of
OTX2, leading to a reduction in the expression of p16, which might contribute to the development of lung cancer.