HOXD1 inhibits lung adenocarcinoma progression and is regulated by DNA methylation [TBS]. HOXD1 inhibits lung adenocarcinoma progression and is regulated by DNA methylation [TBS]

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 investigate that the HOXD1 promoter undergoes DNA methylation, targeted bisulfite sequencing (TBS) was performed to detect methylation levels of HOXD1 in BEAS-2B, A549, and H1299 cells.

同源框（homeobox, HOX）家族编码高度保守的转录因子，在胚胎发育及肿瘤发生过程中发挥关键作用。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，与癌旁正常组织相比，肺腺癌患者样本中HOXD1呈低表达状态。蛋白质印迹（Western blotting）实验验证了肺腺癌（lung adenocarcinoma, LUAD）细胞系中HOXD1的低表达水平。
Kaplan-Meier绘图器（Kaplan-Meier plotter）数据库结果显示，肺腺癌组织中HOXD1表达降低与较差的总生存期显著相关。与此同时，体外研究表明，HOXD1过表达可抑制肺腺癌细胞的增殖、迁移与侵袭能力。在小鼠肿瘤模型中，HOXD1过表达可延缓肿瘤生长进程。
此外，靶向亚硫酸氢盐测序及染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验发现，HOXD1基因启动子区域存在DNA高甲基化现象，且该过程与DNA甲基转移酶相关。作为转录因子的HOXD1过表达可上调骨形态发生蛋白2（bone morphogenetic protein 2, BMP2）与骨形态发生蛋白6（bone morphogenetic protein 6, BMP6）的转录表达水平。
综上，DNA甲基化介导的HOXD1表达失调通过调控BMP2/BMP6的基因表达，抑制了肺腺癌的发生与进展。
实验整体设计：为探究HOXD1启动子的DNA甲基化情况，本研究采用靶向亚硫酸氢盐测序（targeted bisulfite sequencing, TBS）检测BEAS-2B、A549及H1299细胞中HOXD1的甲基化水平。