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Methylated +322-327 CpG site decreases hOGG1 mRNA expression in non-small cell lung cancer
Yuanyuan Zeng1, Jianjie Zhu1, Hualong Qin2
1Department of Respiratory Medicine, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, P.R. China.
Abstract:
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.
Insights
DNA methylation of the hOGG1 gene promoter, specifically at the +322-327 CpG site in the 5'-UTR, is linked to reduced hOGG1 expression in non-small cell lung cancer (NSCLC). This methylation decreases Sp1 transcription factor binding, leading to lower hOGG1 mRNA levels in NSCLC tissues.
Area of Science:
- Molecular Biology
- Cancer Research
- Epigenetics
Background:
- hOGG1 is crucial in DNA repair and implicated in various cancers.
- Transcriptional regulation of hOGG1 in non-small cell lung cancer (NSCLC), particularly via DNA methylation, is not well understood.
- Previous studies suggest frequent promoter methylation of hOGG1 in NSCLC.
Purpose of the Study:
- To investigate the transcriptional regulation of hOGG1 in NSCLC, focusing on DNA methylation.
- To identify specific CpG sites and regulatory elements involved in hOGG1 gene expression changes in NSCLC.
- To elucidate the mechanism by which DNA methylation affects hOGG1 expression and its association with the transcription factor Sp1.
Main Methods:
- Analysis of hOGG1 mRNA expression in NSCLC and adjacent non-cancerous tissues.
- Treatment of NSCLC cells with 5-Aza (a demethylating agent) to assess the impact of DNA methylation on hOGG1 expression.
- MassARRAY EpiTYPER and luciferase reporter gene assays to map functional regions and CpG site methylation.
- Chromatin immunoprecipitation (ChIP) assay to verify transcription factor binding to the hOGG1 5 -UTR.
Main Results:
- hOGG1 mRNA expression was found to be downregulated in NSCLC tissues compared to non-cancerous tissues.
- Demethylation using 5-Aza significantly restored hOGG1 expression in NSCLC cell lines.
- Increased methylation at the +322-327 CpG site in the hOGG1 5 -UTR was observed in NSCLC tissues and inversely correlated with hOGG1 mRNA levels.
- Methylation of this CpG site reduced Sp1 transcription factor binding to the hOGG1 5 -UTR, decreasing transcriptional activity by approximately 50%.
Conclusions:
- hOGG1 mRNA is downregulated in NSCLC, associated with increased DNA methylation at the +322-327 CpG site in its 5 -UTR.
- This methylation event impairs the recruitment of the Sp1 transcription factor, thereby reducing hOGG1 gene expression.
- The findings highlight a novel epigenetic mechanism regulating hOGG1 in NSCLC, offering potential therapeutic targets.
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