The transcription activity of OTX2 on p16 expression is significantly blocked by methylation of CpG shore in non-promoter of lung cancer cell lines

Abstract

Background The aberrant expression of the classical tumor suppressor gene p16 is a frequent event in lung cancer mainly due to the hypermethylation of its 5’-cytosine-phosphate-guanine-3’ island (Cgi). However, whether methylation happens in other regions and how p16 expression and function are affected are largely unknown. Methods Clustered Regularly Interspaced Short Palindromic Repeats/dCas9 (CRISPR/dCas9) technology was used for methylation editing at specific site of p16 . The effects of methylation editing were detected by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt (MTS), transwell migration and wound healing tests. Chromatin immnoprecipitation-quantitative polymerase chain reaction (CHIP-qPCR) was performed to explore the impact of Cgi shore methylation on the binding abilities of transcription factors (TFs) including YY1 , SP1 , ZNF148 and OTX2 to p16 gene. A rescue experiment was performed to verify the regulatory effect of OTX2 on p16 . The negative relationship between p16 expression and the methylation level of Cgi shore in non-promoter region was further verified with datasets from The Cancer Genome Atlas (TCGA) program and lung adenocarcinoma (LUAD) patients’ samples. Results The suppressive effect of p16 Cgi shore methylation on its expression was demonstrated in both HEK293 and A549 cells using CRISPR/dCas9-mediated specific site methylation editing. Methylation of the Cgi shore in the p16 non-promoter region significantly decreased its expression and promoted cell growth and migration. The ability of OTX2 bound to p16 was significantly reduced by 19.35% after methylation modification. Over-expression of OTX2 in A549 cells partly reversed the inhibitory effect of methylation on p16 expression by 19.04%. The verification results with TCGA and LUAD patients’ samples supported that the p16 Cgi shore is a key methylation regulatory region. Conclusions Our findings suggested that methylation of the Cgi shore in the p16 non-promoter region can hamper the transcriptional activity of OTX2 , leading to a reduction in the expression of p16 , which might contribute to the development of lung cancer.

Research progress and applications of epigenetic biomarkers in cancer

“…It functions by inhibiting the growth of tumor-killing cells (Muss et al, 2020). Several studies have indicated that hypermethylation of the p16 gene is strongly associated with poor prognosis in patients diagnosed with colorectal cancer (Karam et al, 2019), liver cancer (Wong et al, 1999), and lung cancer (Peng et al, 2023). Another gene involved in cancer development is glutathione S-transferase 1 (GSTP1).…”