Zeng Yuanyuan, Zhu Jianjie, Qin Hualong, Shen Dan, Lei Zhe, Li Wei, Ding Zongli, Huang Jian-An, Liu Zeyi
Department of Respiratory Medicine, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, P.R. China.
Department of Cardiothoracic surgery, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, P.R. China.
Oncol Rep. 2017 Jul;38(1):529-537. doi: 10.3892/or.2017.5690. Epub 2017 Jun 1.
hOGG1 plays a role in several disease pathways, including various cancers. Despite such functional importance, how hOGG1 is regulated at the transcriptional level in human non-small cell lung cancer (NSCLC) remains unknown, particularly via DNA methylation changes. We obtained NSCLC tissues and adjacent non-cancerous tissues and examined hOGG1 mRNA expression levels. NSCLC cells were treated with 5-Aza to test whether DNA methylation can influence the expression of hOGG1. The MassARRAY EpiTYPER and luciferase reporter gene assays were used to define the functional region of the hOGG1 gene (including CpG sites). Finally, ChIP assay was utilized to verify transcription factor binding to the hOGG1 5'-UTR region. Our previous studies supported the idea that the methylation of the hOGG1 gene promoter region occurs frequently in NSCLC. Treatment with 5-Aza, a demethylating agent, led to a significant restoration of hOGG1 expression in NSCLC cell lines. Quantitative PCR and MassARRAY EpiTYPER assays demonstrated that methylation of the +322-327 CpG site in the 5'-UTR region of hOGG1 was higher in NSCLC tissues compared with adjacent non-cancerous tissues. Notably, the methylation level of +322-327 site (T/N) was inversely correlated with that of hOGG1 mRNA level (T/N) in 25 NSCLC tissues. ChIP assay and in silico prediction showed an association between the +322-327 CpG site and Sp1, which has been reported to be an activator of transcription. Importantly, luciferase reporter gene and ChIP assays showed that +322-327 CpG site methylation particularly reduced the recruitment of Sp1 to the 5'-UTR sequence in hOGG1 and reduced transcriptional activity ~50%. In summary, we have demonstrated that hOGG1 mRNA is downregulated in NSCLC tissues. Moreover, we identified that the methylated +322-327 CpG site in the hOGG1 5'-UTR is associated with reduced expression of hOGG1 by decreasing the recruitment of Sp1 to the 5'-UTR of hOGG1.
hOGG1在包括各种癌症在内的多种疾病途径中发挥作用。尽管具有如此重要的功能，但hOGG1在人类非小细胞肺癌（NSCLC）中在转录水平是如何被调控的，尤其是通过DNA甲基化变化来调控，仍然未知。我们获取了NSCLC组织和相邻的非癌组织，并检测了hOGG1 mRNA表达水平。用5-氮杂胞苷处理NSCLC细胞，以测试DNA甲基化是否会影响hOGG1的表达。使用MassARRAY EpiTYPER和荧光素酶报告基因检测来确定hOGG1基因的功能区域（包括CpG位点）。最后，利用染色质免疫沉淀（ChIP）检测来验证转录因子与hOGG1 5'-非翻译区（UTR）区域的结合。我们之前的研究支持这样的观点，即hOGG1基因启动子区域的甲基化在NSCLC中频繁发生。用去甲基化剂5-氮杂胞苷处理导致NSCLC细胞系中hOGG1表达显著恢复。定量PCR和MassARRAY EpiTYPER检测表明，与相邻的非癌组织相比，NSCLC组织中hOGG1 5'-UTR区域中+322 - 327 CpG位点的甲基化程度更高。值得注意的是，在25个NSCLC组织中，+322 - 327位点（T/N）的甲基化水平与hOGG1 mRNA水平（T/N）呈负相关。ChIP检测和计算机模拟预测显示+322 - 327 CpG位点与Sp1之间存在关联，Sp1已被报道为一种转录激活因子。重要的是，荧光素酶报告基因和ChIP检测表明，+322 - 327 CpG位点甲基化特别降低了Sp1对hOGG1中5'-UTR序列的募集，并使转录活性降低了约50%。总之，我们已经证明hOGG1 mRNA在NSCLC组织中表达下调。此外，我们发现hOGG1 5'-UTR中甲基化的+322 - 327 CpG位点通过减少Sp1对hOGG1 5'-UTR的募集而与hOGG1表达降低相关。
