Liao Cheng-Gong, Liang Xiao-Hua, Ke Yuan, Yao Li, Liu Man, Liu Ze-Kun, He Lin, Guo Yi-Xiao, Bian Huijie, Chen Zhi-Nan, Kong Ling-Min
Department of Oncology, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, People's Republic of China.
Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, 710038, People's Republic of China.
Oncogene. 2022 Mar;41(12):1780-1794. doi: 10.1038/s41388-022-02213-0. Epub 2022 Feb 7.
Non-small cell lung cancer (NSCLC) is a fatal disease, and its metastatic process is poorly understood. Although aberrant methylation is involved in tumor progression, the mechanisms underlying dynamic DNA methylation remain to be elucidated. It is significant to study the molecular mechanism of NSCLC metastasis and identify new biomarkers for NSCLC early diagnosis. Here, we performed MeDIP-seq and hMeDIP-seq analyses to detect the genes regulated by dynamic DNA methylation. Comparison of the 5mC and 5hmC sites revealed that the CD147 gene underwent active demethylation in NSCLC tissues compared with normal tissues, and this demethylation upregulated CD147 expression. Significantly high levels of CD147 expression and low levels of promoter methylation were observed in NSCLC tissues. Then, we identified the CD147 promoter as a target of KLF6, MeCP2, and DNMT3A. Treatment of cells with TGF-β triggered active demethylation involving loss of KLF6/MeCP2/DNMT3A and recruitment of Sp1, Tet1, TDG, and SMAD2/3 transcription complexes. A dCas9-SunTag-DNMAT3A-sgCD147-targeted methylation system was constructed to reverse CD147 expression. The targeted methylation system downregulated CD147 expression and inhibited NSCLC proliferation and metastasis in vitro and in vivo. Accordingly, we used cfDNA to detect the levels of CD147 methylation in NSCLC tissues and found that the CD147 methylation levels exhibited an inverse relationship with tumor size, lymphatic metastasis, and TNM stage. In conclusion, this study clarified the mechanism of active demethylation of CD147 and suggested that the targeted methylation of CD147 could inhibit NSCLC invasion and metastasis, providing a highly promising therapeutic target for NSCLC.
非小细胞肺癌（NSCLC）是一种致命疾病，其转移过程尚不清楚。虽然异常甲基化参与肿瘤进展，但动态DNA甲基化的潜在机制仍有待阐明。研究NSCLC转移的分子机制并鉴定NSCLC早期诊断的新生物标志物具有重要意义。在此，我们进行了甲基化DNA免疫沉淀测序（MeDIP-seq）和羟甲基化DNA免疫沉淀测序（hMeDIP-seq）分析，以检测受动态DNA甲基化调控的基因。5-甲基胞嘧啶（5mC）和5-羟甲基胞嘧啶（5hmC）位点的比较显示，与正常组织相比，NSCLC组织中CD147基因发生了主动去甲基化，这种去甲基化上调了CD147的表达。在NSCLC组织中观察到CD147表达水平显著升高，启动子甲基化水平降低。然后，我们确定CD147启动子是KLF6、甲基CpG结合蛋白2（MeCP2）和DNA甲基转移酶3A（DNMT3A）的靶点。用转化生长因子-β（TGF-β）处理细胞会引发主动去甲基化，涉及KLF6/MeCP2/DNMT3A的缺失以及Sp1、Tet1、胸腺嘧啶DNA糖基化酶（TDG）和SMAD2/3转录复合物的募集。构建了一种靶向甲基化系统，即dCas9-SunTag-DNMAT3A-sgCD147，以逆转CD147的表达。该靶向甲基化系统下调了CD147的表达，并在体外和体内抑制了NSCLC的增殖和转移。因此，我们使用游离DNA（cfDNA）检测NSCLC组织中CD147的甲基化水平，发现CD147甲基化水平与肿瘤大小、淋巴转移和TNM分期呈负相关。总之，本研究阐明了CD147主动去甲基化的机制，并表明CD147的靶向甲基化可抑制NSCLC的侵袭和转移，为NSCLC提供了一个极有前景的治疗靶点。
