HOXD1 inhibits lung adenocarcinoma progression and is regulated by DNA methylation [ChIP-seq]. HOXD1 inhibits lung adenocarcinoma progression and is regulated by DNA methylation [ChIP-seq]

The homeobox (HOX) family encodes highly conserved transcription factors and plays a crucial role in embryonic development and tumorigenesis. Homeobox D1 (HOXD1) is a member of the HOX family, whose biological functions in lung cancer are unclear. The UALCAN database analysis of HOXD1 expression patterns revealed that HOXD1 is downregulated in lung adenocarcinoma patient samples relative to adjacent normal tissue. Western blotting validated low HOXD1 expression in lung adenocarcinoma (LUAD) cell lines. The Kaplan-Meier plotter database demonstrated that HOXD1 expression reduction in LUAD was found to correlate with worse overall survival. Meanwhile, in vitro study showed that HOXD1 overexpression suppressed LUAD cells proliferation, migration, and invasion. In the mouse tumor model, upregulated HOXD1 retarded tumor growth. In addition, targeted bisulfite sequencing and ChIP assay found that DNA hypermethylation occurred in the promoter region of the HOXD1 gene and was associated with DNA methyltransferases. In additionMoreover, upregulated HOXD1 as a transcriptional factor increased the transcriptional expression of BMP2 and BMP6. Taken together, the dysregulation of HOXD1 mediated by DNA methylation inhibited the initiation and progression of lung adenocarcinoma by regulating the expression of BMP/BMP6. Overall design: To better identify the downstream target gene potentially regulated by HOXD1 as a transcription factor in LUAD, ChIP-seq was performed on HOXD1-overexpression A549 cells to analyze the downstream regulatory network of HOXD1.

同源框（homeobox, HOX）家族编码高度保守的转录因子，在胚胎发育与肿瘤发生过程中发挥关键调控作用。同源框D1（homeobox D1, HOXD1）为HOX家族成员之一，其在肺癌中的生物学功能尚未明确。通过UALCAN数据库分析HOXD1的表达模式发现，相较于癌旁正常组织，肺腺癌（lung adenocarcinoma, LUAD）患者样本中HOXD1呈现表达下调趋势。蛋白质印迹（Western blotting）实验验证了LUAD细胞系中HOXD1的低表达水平。卡普兰-迈耶绘图器（Kaplan-Meier plotter）数据库分析显示，LUAD组织中HOXD1表达降低与患者较差的总生存期显著相关。与此同时，体外实验证实HOXD1过表达可抑制LUAD细胞的增殖、迁移及侵袭能力。在小鼠肿瘤模型中，HOXD1上调能够延缓肿瘤生长。此外，靶向亚硫酸氢盐测序（targeted bisulfite sequencing）与染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验发现，HOXD1基因启动子区域存在DNA高甲基化现象，且该修饰与DNA甲基转移酶存在关联。进一步研究显示，作为转录因子的HOXD1过表达可上调骨形态发生蛋白2（BMP2）与骨形态发生蛋白6（BMP6）的转录水平。综上，由DNA甲基化介导的HOXD1表达失调通过调控BMP/BMP6通路的基因表达，抑制了肺腺癌的发生与进展。整体实验设计：为精准鉴定HOXD1作为转录因子在LUAD中潜在调控的下游靶基因，本研究对HOXD1过表达的A549细胞开展染色质免疫沉淀测序（ChIP-seq），以解析HOXD1的下游调控网络。